Composition and method for treating inflammatory disorders

ABSTRACT

Inhibitors of IL-22 are disclosed as well as pharmaceutical compositions and methods of using same. The inhibitors include IL-22 antibodies and are useful for treating inflammatory disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 60/270,823, filed Feb.23, 2001; and to U.S. Ser. No. 60/281,353, filed Apr. 3, 2001. Thepresent application also claims priority to U.S. Ser. No. 09/561,811,filed Apr. 28, 2000, which claims the benefit of prior-filed provisionalapplication U.S. Ser. No. 60/131,473, filed Apr. 28, 1999. The contentsof all of these patent applications are incorporated herein by thisreference in their entirety.

FIELD OF THE INVENTION

The invention is related to cytokine polypeptides and polynucleotidesencoding these polypeptides. The invention is also related to antibodiesto cytokine polypeptides and to therapeutic compositions that includethese antibodies. The compositions are useful in treating, e.g.,inflammatory states such as arthritis.

BACKGROUND OF THE INVENTION

Inflammatory arthritis represents is a family of arthritic diseasescharacterized by lymphokine-mediated inflammation of the joints.Inflammatory arthritis is often autoimmune in origin. Examples ofinflammatory arthritis can include rheumatoid arthritis, psoriaticarthritis, and lupus-associated arthritis. The most common form ofinflammatory arthritis is rheumatoid arthritis. Rheumatoid arthritis ischaracterized by persistent inflammation of the joints. Inflammation caneventually lead to cartilage destruction and bone erosion.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that inhibitors of thecytokine IL-22 can inhibit symptoms associated with arthritis. Moreparticularly, antibodies raised against IL-22 (which is also referred toherein as GIL-19 and AE289) have been found to inhibit the developmentof symptoms associated with collagen-induced arthritis in a murine modelsystem.

In one aspect, the invention features an antibody which immunologicallyreacts with an IL 22 protein. The antibody can be, e.g., a neutralizingantibody. In preferred embodiments, the antibody is a monoclonalantibody. Examples of monoclonal antibodies include a human monoclonalantibody and a humanized monoclonal antibody.

In some embodiments, the antibody binds specifically to a polypeptidethat includes the amino acid sequence of SEQ ID NO:2, which correspondsto the amino acid sequence of a human IL-22 polypeptide.

Also provided by the invention is a pharmaceutical compositioncomprising an IL-22 antibody which immunologically reacts with an IL 22protein comprising the amino acid sequence of SEQ ID NO:2 and apharmaceutical carrier. The antibody in the pharmaceutical compositionis preferably a neutralizing antibody. The antibody is preferably amonoclonal antibody, such as a human monoclonal antibody or a humanizedmonoclonal antibody.

Also provided by the invention is a method of treating a pathologicalcondition in a subject associated with IL-22 activity by administeringan effective amount of an agent that inhibits levels of IL-22 activity,thereby treating the pathological condition.

The pathological condition can be, e.g., septicemia, and autoimmunedisorders. Suitable autoimmune disorders include, e.g., rheumatoidarthritis, osteoarthritis, multiple sclerosis, myasthenia gravis,inflammatory bowel disease, lupus, diabetes and psoriasis. Theseresponses can be associated wound healing processes, cholesterolmetabolism, oxygen free radical injury, ischemia, atherosclerosis andallergies.

Also provided by the invention is a method of treating symptomsassociated with arthritis, the method by administering to a subject inneed thereof a therapeutically effective amount of an IL-22 antibody. Insome embodiments, the arthritis is rheumatoid arthritis.

The IL-22 antibody can be administered therapeutically orprophylactically, or both.

Also provided by the invention is a method of enhancing a subject'simmune response to an antigen by administering to the subject animmunogenic amount of the antigen and an immunogenicity-augmentingamount of IL-22 in concurrent or sequential combination with the antigensuch that the subject's immune response is enhanced.

Also provided is a method of treating a pathological condition in asubject in need of IL-22 modulation by administering an effective amountof an agent that modulates IL-22 activity, such that the pathologicalcondition in the subject is treated.

The work described herein reveals that IL-22 is a cytokine involved inacute phase responses. The invention provides for the use of IL-22 aswell as IL-22 modulatory agents (i.e., agents that stimulate or inhibitIL-22 activity) to alter an immune response, either through itsupregulation or down-regulation, depending on the clinical situation.

As used herein, the term “IL-22” molecule includes nucleic acidmolecules, proteins, polypeptides, and fragments or variants thereofhaving at least one IL-22 activity as defined herein. In a preferredembodiment, the IL-22 molecule is a human IL-22 molecule (e.g. the humanIL-22 nucleic acid and protein molecules set forth in SEQ ID NO: 1 andSEQ ID NO:2).

In one embodiment, the present invention provides a compositioncomprising an isolated polynucleotide selected from the group consistingof:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:1;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:1 from nucleotide 65 to nucleotide 601;    -   (c) a polynucleotide comprising the nucleotide sequence of the        full-length protein coding sequence of clone IL-22 deposited        under accession number ATCC 207231;    -   (d) a polynucleotide encoding the full-length protein encoded by        the cDNA insert of clone IL-22 deposited under accession number        ATCC 207231;    -   (e) a Polynucleotide comprising the nucleotide sequence of a        mature protein coding sequence of clone IL-22 deposited under        accession number ATCC 207231;    -   (f) a polynucleotide encoding a mature protein encoded by the        cDNA insert of clone IL-22 deposited under accession number ATCC        207231;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:2;    -   (h) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:2 having biological        activity, the fragment comprising eight contiguous amino acids        of SEQ ID NO:2;    -   (i) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(f) above;    -   (j) a polynucleotide which encodes a species homologue of the        protein of (g) or (h) above;    -   (k) a polynucleotide that hybridizes under stringent conditions        to any one of the polynucleotides specified in (a)-(h); and

a polynucleotide that hybridizes under stringent conditions to any oneof the polynucleotides specified in (a)-(h) and that has a length thatis at least 25% of the length of SEQ ID NO:1.

Preferably, such polynucleotide comprises the nucleotide sequence of SEQED NO:1 from nucleotide 65 to nucleotide 601; the nucleotide sequence ofthe full-length protein coding sequence of clone IL-22 deposited underaccession number ATCC 207231; or the nucleotide sequence of a matureprotein coding sequence of clone IL-22 deposited under accession numberATCC 207231 (e.g., nucleotides 1-1177 of SEQ ID NO:1). In otherpreferred embodiments, the polynucleotide encodes the full-length or amature protein encoded by the cDNA insert of clone IL-22 deposited underaccession number ATCC 207231 (e.g., amino acids 1-179 of SEQ ID NO: 2).In further preferred embodiments, the present invention provides apolynucleotide encoding a protein comprising a fragment of the aminoacid sequence of SEQ ID NO:2 having biological activity, the fragmentpreferably comprising eight (more preferably twenty, most preferablythirty) contiguous amino acids of SEQ ID NO:2, or a polynucleotideencoding a protein comprising a fragment of the amino acid, sequence ofSEQ ID NO:2 having biological activity, the fragment comprising theamino acid sequence from amino acid 84 to amino acid 93 of SEQ ID NO:2.

Other embodiments provide the gene corresponding to the cDNA sequence ofSEQ ID NO:1.

Further embodiments of the invention provide isolated polynucleotidesproduced according to a process selected from the group consisting of:

-   -   (a) a process comprising the steps of:        -   (i) preparing one or more polynucleotide probes that            hybridize in 6×SSC at 65 degrees C. to a nucleotide sequence            selected from the group consisting of:            -   (aa) SEQ ID NO:1, but excluding the poly(A) tail at the                3′ end of SEQ ID NO:1; and            -   (ab) the nucleotide sequence of the cDNA insert of clone                IL-22 deposited under accession number ATCC 207231;        -   (ii) hybridizing said probe(s).to human genomic DNA in            conditions at least as stringent as 4×SSC at 50 degrees C.;            and        -   (iii) isolating the DNA polynucleotides detected with the            probe(s); and    -   (b) a process comprising the steps of:        -   (i) preparing one or more polynucleotide primers that            hybridize in 6×SSC at 65 degrees C. to a nucleotide sequence            selected from the group consisting of:        -   (ba) SEQ ID NO:1, but excluding the poly(A) tail at the 3′            end of SEQ ID NO:1; and        -   (bb) the nucleotide sequence of the cDNA insert of clone            IL-22 deposited under accession number ATCC 207231;    -   (ii) hybridizing said primer(s) to human genomic DNA in        conditions at least as stringent as 4×SSC at 50 degrees C.;    -   (iii) amplifying human DNA sequences; and    -   (iv) isolating the polynucleotide products of step (b)(iii).

Preferably the polynucleotide isolated according to the above processcomprises a nucleotide sequence corresponding to the cDNA sequence ofSEQ ID NO:1, and extending contiguously from a nucleotide sequencecorresponding to the 5′ end of SEQ ID NO:1 to a nucleotide sequencecorresponding to the 3′ end of SEQ ID NO:1, but excluding the poly(A)tail at the 3′ end of SEQ ID NO:1. Also preferably the polynucleotideisolated according to the above process comprises a nucleotide sequencecorresponding to the cDNA sequence of SEQ ID NO:1 from nucleotide 65 tonucleotide 601, and extending contiguously from a nucleotide sequencecorresponding to the 5′ end of said sequence of SEQ ID NO:1 fromnucleotide 65 to nucleotide 601, to a nucleotide sequence correspondingto the 3′ end of said sequence of SEQ ID NO:1 from nucleotide 65 tonucleotide 601.

In other embodiments, the present invention provides a compositioncomprising a protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) a fragment of the amino acid sequence of SEQ ID NO:2, the        fragment comprising eight contiguous amino acids of SEQ ID NO:2;        and    -   (c) the amino acid sequence encoded by the cDNA insert of clone        IL-22 deposited under accession number ATCC 207231;        the protein being substantially free from other mammalian        proteins. Preferably such protein comprises the amino acid        sequence of SEQ ID NO:2. In further preferred embodiments, the        present invention provides a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:2 having biological        activity, the fragment preferably comprising eight (more        preferably twenty, most preferably thirty) contiguous amino        acids of SEQ ID NO:2.

In certain preferred embodiments, the polynucleotide is operably linkedto an expression control sequence. The invention also provides a hostcell, including bacterial, yeast, insect and mammalian cells,transformed with such polynucleotide compositions. Also provided by thepresent invention are organisms that have enhanced, reduced, or modifiedexpression of the gene(s) corresponding to the polynucleotide sequencesdisclosed herein.

Processes are also provided for producing a protein, which comprise:

-   -   (a) growing a culture of the host cell transformed with such        polynucleotide compositions in a suitable culture medium; and    -   (b) purifying the protein from the culture.        The protein produced according to such methods is also provided        by the present invention.

Protein compositions of the present invention may further comprise apharmaceutically acceptable carrier. Compositions comprising an antibodywhich specifically reacts with such protein are also provided by thepresent invention.

As used herein, a “modulator”, “agent” or “modulating agent” includesany proteins, polypeptides, nucleic acids, agonists or antagonists orprotein, polypeptide or nucleic acid fragments or variants thereof thatare capable of altering the activity of IL-22. For example, suchaltering of IL-22 activity may include the blocking of IL-22 activity,down-regulation of IL-22 activity or inhibition of IL-22 activity.Alternatively, the alteration of IL-22 activity may include theaugmenting of IL-22 activity, the up-regulation of IL-22 activity or theenhancing of IL-22 activity.

In another embodiment, modulators that act on the present invention areprovided. In a preferred embodiment, polyclonal and/or monoclonalantibodies of the present invention (e.g. neutralizing antibody specificfor IL-22) are also provided to down-modulate an immune responses (i.e.to treat sepsis and other chronic inflammatory disorders). In anotherpreferred embodiment, the present invention is used as a vaccineadjuvant to alter the type of immune response achieved by antigen alone.

In another embodiment, a method of treating a pathological condition ina subject is provided by modulating the activity of IL-22, such that thepathological condition is treated. In a preferred embodiment, thepathological condition treated is an infectious disease. Morepreferably, the infectious disease can be initiated by a bacteria,virus, parasite or fungi. In another preferred embodiment, thepathological condition is cancer, more preferably renal cell carcinoma.

In another embodiment, modulatory agents are used to alter inflammatorypathologies in the kidney, such as the induction of renal proximaltubular basophilia.

In another embodiment, the present invention will be used for theremodeling of tissues, both in vivo and ex vivo. In a preferredembodiment, IL-22, or agonists of IL-22 is used in the remodeling ofepithelial tissue in the kidney.

In another embodiment, a method is provided for studying a disease in asubject comprising administering an agent that modulates the activity ofIL-22 in vivo, such that the disease can be studied. In a preferredembodiment, the subject under study is a genetically altered mammal,preferably a genetically altered mouse, most preferably a transgenic orgene knock-out mouse.

Methods are also provided for preventing, treating or ameliorating amedical condition which comprises administering to a mammalian subject atherapeutically effective amount of a composition comprising a proteinof the present invention and a pharmaceutically acceptable carrier.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing an experimental protocol used toanalyze the effect of an IL-22 antibody on an in vivo murine arthritismodel.

FIG. 2 is a graph showing body score following treatment of arthriticmice with IL-22 antibody or control using a therapeutic treatmentregimen.

FIG. 3 is a graph showing body score following treatment of arthriticmice with IL-22 antibody or control using a prophylactic treatmentregimen.

FIG. 4 is a graph showing body score following treatment of severelyarthritic mice with IL-22 antibody or control.

FIG. 5 is graph showing relative percentages of paws showing a givenhistology grade following with IL-22 antibody or control.

DETAILED DESCRIPTION

The invention provides inhibitors of IL-22 and therapeutic compositionsthat include the antibodies. The inhibitors are useful for treatinginflammatory states, which include, e.g., autoimmune diseases such asrheumatoid arthritis.

Inhibitors can be prepared using IL-22 polypeptide sequences and nucleicacids encoding same. IL-22 is produced by activated human and mouse Th1,but not Th2, CD4⁺ cells. The cytokine is a multifunctional moleculewhose expression is stimulated by LPS, but not IFN-γ. IL-22 sharesapproximately 20% homology with IL-10 and is produced by activated humanand mouse Th1, but not Th2, CD4⁺ cells. Furthermore, LPS, but not IFN-γ,strongly stimulates IL-22 production from the adherent cell compartmentof murine PECs, indicating that IL-22 is involved in mediating naturalimmunity.

The addition of either an adenovirus encoding murine IL-22 orrecombinant purified murine IL-22 intravenously injected into C57b/6mice induced numerous systemic effects, including decreased red bloodcell count, increased platelet count, decreased serum albumin, increasedserum amyloid A and fibrinogen levels, and decreased body weight, allsuggestive of an acute phase reaction. Moreover, IL-22 administrationalso induces basophilia in the proximal renal tubules, a findingdistinct from an acute phase reaction, suggestive of induced cellularproliferation. Identification of these biological activities of IL-22has led to the development of new approaches to and therapeutics usefulfor the treatment of various immune response-related diseases anddisorders. Moreover, the role of IL-22 in processes including sepsis,chronic inflammation, and autoimmunity has been analyzed and newmechanisms for treating such conditions is disclosed herein.

I. Isolated IL-22 Proteins and Polynucleotides

IL-22 nucleotide and amino acid sequences are provided below. Thenucleotide sequence of each clone can also be determined by sequencingof the deposited clone in accordance with known methods. The predictedamino acid sequence (both full-length and mature forms) can then bedetermined from such nucleotide sequence. The amino acid sequence of theprotein encoded by a particular clone can also be determined byexpression of the clone in a suitable host cell, collecting the proteinand determining its sequence.

As used herein a “secreted” protein is one which, when expressed in asuitable host cell, is transported across or through a membrane,including transport as a result of signal sequences in its amino acidsequence. “Secreted” proteins include without limitation proteinssecreted wholly (e.g., soluble proteins) or partially (e.g., receptors)from the cell in which they are expressed. “Secreted” proteins alsoinclude without limitation proteins which are transported across themembrane of the endoplasmic reticulum.

A polynucleotide of the present invention has been identified initiallyas clone “hTIF/AE289”, later renamed and referred to herein also as“IL-22.” Clone IL-22 was isolated according to the following method. Amurine EST was identified from a murine cDNA library made fromsplenocytes activated with both ConA and bone marrow derived dendriticcells. The EST was identified using methods which are selective forcDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637). Themurine EST sequence was used to isolate a full-length murine clone fromthe same cDNA library (SEQ ID NO:3, shown below). Analysis of thesequence of the murine clone revealed a significant homology tointerleukin-10 (IL-10).

In order to isolate a human homolog of the murine clone, PCR primerswere constructed based upon the region of the murine sequence whichshowed homology to IL-10. Use of such primers for amplification in cDNAlibrary derived from PHA/PMA stimulated human PBMCs produced a PCRproduct of significant size. Analysis of the sequence of the PCR productconfirmed that it was a homolog of the murine cDNA. Oligonucleotideswere constructed from the sequence of the partial human clone and usedto isolate a full-length human clone from the PBMC library.

The disclosed human IL-22 nucleotide sequence is a full-length clone,including the entire coding sequence of a secreted protein. Analysis ofits sequence confirms its homology to IL-10 at a level of 20%.

The nucleotide sequence of the disclosed human IL-22 polynucleotidesequence is reported below (SEQ ID NO:1), and includes a poly(A) tail.The disclosed nucleotide sequence includes an open reading frame and theamino acid sequence of full-length IL-22 protein corresponding to theforegoing nucleotide sequence is reported in SEQ ID NO:2. The amino acidsequence of mature IL-22 corresponds to amino acids 34-179 of SEQ IDNO:2.

(SEQ ID NO: 1) 1 GAATTCGGCC AAAGAGGCCT ACAGGTTCTC CTTCCCCAGT CACCAGTTGC51 TCGAGTTAGA ATTGTCTGCA ATGGCCGCCC TGCAGAAATC TGTGAGCTCT 201TTCCTTATGG GGACCCTGGC CACCAGCTGC CTCCTTCTCT TGGCCCTCTT 151GGTACAGGGA GGAGCAGCTG CGCCCATCAG CTCCCACTGC AGGCTTGACA 201AGTCCAACTT CCAGCAGCCC TATATCACCA ACCGCACCTT CATGCTGGCT 251AAGGAGGCTA GCTTGGCTGA TAACAACACA GACGTTCGTC TCATTGGGGA 301GAAACTGTTC CACGGAGTCA GTATGAGTGA GCGCTGCTAT CTGATGAAGC 351AGGTGCTGAA CTTCACCCTT GAAGAAGTGC TGTTCCCTCA ATCTGATAGG 401TTCCAGCCTT ATATGCAGGA GGTGGTGCCC TTCCTGGCCA GGCTCAGCAA 451CAGGCTAAGC ACATGTCATA TTGAAGGTGA TGACCTGCAT ATCCAGAGGA 501ATGTGCAAAA GCTGAAGGAC ACAGTGAAAA AGCTTGGAGA GAGTGGAGAG 551ATCAAAGCAA TTGGAGAACT GGATTTGCTG TTTATGTCTC TGAGAAATGC 601CTGCATTTGA CCAGAGCAAA GCTGAAAAAT GAATAACTAA CCCCCTTTCC 651CTGCTAGAAA TAACAATTAG ATGCCCCAAA GCGATTTTTT TTAACCAAAA 701GGAAGATGGG AAGCCAAACT CCATCATGAT GGGTGGATTC CAAATGAACC 751CCTGCGTTAG TTACAAAGGA AACCAATGCC ACTTTTGTTT ATAAGACCAG 801AAGGTAGACT TTCTAAGCAT AGATATTTAT TGATAACATT TCATTGTAAC 851TGGTGTTCTA TACACAGAAA ACAATTTATT TTTTAAATAA TTGTCTTTTT 901CCATAAAAAA GATTACTTTC CATTCCTTTA GGGGAAAAAA CCCCTAAATA 951GCTTCATGTT TCCATAATCA GTACTTTATA TTTATAAATG TATTTATTAT 1001TATTATAAGA CTGCATTTTA TTTATATCAT TTTATTAATA TGGATTTATT 1051TATAGAAACA TCATTCGATA TTGCTACTTG AGTGTAAGGC TAATATTCAT 1101ATTTATGACA ATAATTATAG AGCTATAACA TGTTTATTTG ACCTCAATAA 1151ACACTTGGAT ATCCTAAAAA AAAAAAAAAA AAAGCGGCCG C

The polypeptide sequence of the encoded polypeptide is shown below.

(SEQ ID NO: 2) 1 MAALQKSVSS FLMGTLATSC LLLLALLVQG GAAAPISSHC RLDKSNFQQP51 YITNRTFMLA KEASLADNNT DVRLIGEKLF HGVSMSERCY LMKQVLNFTL 101EEVLFPQSDR FQPYMQEVVP FLARLSNRLS TCHIEGDDLH IQRNVQKLKD 151TVKKLGESGE IKAIGELDLL FMSLRNACIClone “IL-22” was deposited on Apr. 28, 1999 with the American Type.Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209U.S.A.) as an original deposit under the Budapest Treaty and were giventhe accession number ATCC 207231. All restrictions on the availabilityto the public of the deposited material will be irrevocably removed uponthe granting of the patent, except for the requirements specified in 37C.F.R. §1.808(b), and the term of the deposit will comply with 37 C.F.R.§1.806.

Nucleotide sequences encoding murine IL-22, and the sequence of theencoded polypeptide, are provided below.

(SEG ID NO: 3) 1 GAATTCGGCC AAAGAGGCCT ACCTAAACAG GCTCTCCTCT CAGTTATCAA51 CTGTTGACAC TTGTGCGATC TCTGATGGCT GTCCTGCAGA AATCTATGAG l01TTTTTCCCTT ATGGGGACTT TGGCCGCCAG CTGCCTGCTT CTCATTGCCC 151TGTGGGCCCA GGAGGCAAAT GCGCTGCCCG TCAACACCCG GTGCAAGCTT 201GAGGTGTCCA ACTTCCAGCA GCCATACATC GTCAACCGCA CCTTTATGCT 251GGCCAAGGAG GCCAGCCTTG CAGATAACAA CACAGATGTC CGGCTCATCG 301GGGAGAAACT GTTCCGAGGA GTCAGTGCTA AGGATCAGTG CTACCTGATG 351AAGCAGGTGC TCAACTTCAC CCTGGAAGAC GTTCTGCTCC CCCAGTCAGA 401CAGGTTCCAG CCCTACATGC AGGAGGTGGT GCCTTTCCTG ACCAAACTCA 451GCAATCAGCT CAGCTCCTGT CACATCAGCG GTGACGACCA GAACATCCAG 501AAGAATGTCA GAAGGCTGAA GGAGACAGTG AAAAAGCTTG GAGAGAGTGG 551AGAGATCAAG GCGATTGGGG AACTGGACCT GCTGTTTATG TCTCTGAGAA 601ATGCTTGCGT CTGAGCGAGA AGAAGCTAGA AAACGAAGAA CTGCTCCTTC 651CTGCCTTCTA AAAAGAACAA TAAGATCCCT GAATGGACTT TTTTACTAAA 701GGAAAGTGAG AAGCTAACGT CCATCATTAT TAGAAGATTT CACATGAAAC 751CTGGCTCAGT TGAAAAAGAA AATAGTGTCA AGTTGTCCAT GAGACCAGAG 801GTAGACTTGA TAACCACAAA GATTCATTGA CAATATTTTA TTGTCACTGA 851TGATACAACA GAAAAATAAT GTACTTTAAA AAATTGTTTG AAAGGAGGTT 901ACCTCTCATT CCTTTAGAAA AAAAGCTTAT GTAACTTCAT TTCCATAACC 951AATATTTTAT ATATGTAAGT TTATTTATTA TAAGTATACA TTTTATTTAT 1001GTCAGTTTAT TAATATGGAT TTATTTATAG AAACATTATC TGCTATTGAT 1051ATTTAGTATA AGGCAAATAA TATTTATGAC AATAACTATG GAAACAAGAT 1101ATCTTAGGCT TTAATAAACA CATGGATATC ATAAAAAAAA AAAAAAAAAA 1151AAAAAAAAGC GGCCGC

The amino acid sequence of the polypeptide encoded by theabove-referenced polynucleotide sequence is provide below:

(SEG ID NO: 4) 1 MAVLQKSMSF SLMGTLAASC LLLIALWAQE ANALPVNTRC ELEVSNFQQP51 YIVNRITMLA KEASLADNNT DVRLIGEKLF RGVSAKDQCY LMKQVLNFTL 101EDVLLPQSDR PQPYMQEVVP PLTKLSNQLS SCRISGDDQN IQKNVRRLKE 151TVKKLGESGE IKAIGELDLL FMSLRNACV*

Fragments of an IL-22 protein (e.g. fragments which are capable ofexhibiting biological activity) are also encompassed by the compositionsand methods of the invention. Fragments of the protein can be in linearform, or they can be cyclized using known methods, for example, asdescribed in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992)and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253(1992), both of which are incorporated herein by reference. Suchfragments can be fused to carrier molecules such as immunoglobulins formany purposes, including increasing the valency of protein bindingsites. For example, fragments of the protein can be fused through“linker” sequences to the Fc portion of an immunoglobulin. For abivalent form of the protein, such a fusion can be to the Fc portion ofan IgG molecule. Other immunoglobulin isotypes may also be used togenerate such fusions. For example, a protein-IgM fusion generates adecavalent form of the protein of the invention.

The present invention also provides both full-length and mature forms ofthe disclosed proteins. The full-length form of such proteins isidentified in the sequence listing by translation of the nucleotidesequence of each disclosed clone. The mature form(s) of such protein canbe obtained by expression of the disclosed full-length polynucleotide(preferably those deposited with ATCC) in a suitable mammalian cell orother host cell. The sequence(s) of the mature form(s) of the proteincan also be determined from the amino acid sequence of the full-lengthform. An example of a mature IL-22 polypeptide sequence is amino acids1-179 of SEQ ID NO:2.

The present invention also provides genes corresponding to thepolynucleotide sequences disclosed herein. “Corresponding genes” are theregions of the genome that are transcribed to produce the mRNAs fromwhich cDNA polynucleotide sequences are derived and may includecontiguous regions of the genome necessary for the regulated expressionof such genes. Corresponding genes may therefore include but are notlimited to coding sequences, 5′ and 3′ untranslated regions,alternatively spliced exons, introns, promoters, enhancers, and silenceror suppressor elements. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials. An “isolated gene” is a gene that has beenseparated from the adjacent coding sequences, if any, present in thegenome of the organism from which the gene was isolated.

The chromosomal location corresponding to the polynucleotide sequencesdisclosed herein may also be determined, for example by hybridizingappropriately labeled polynucleotides of the present invention tochromosomes in situ. The corresponding chromosomal location for adisclosed polynucleotide can be determined by identifying significantlysimilar nucleotide sequences in public databases, such as expressedsequence tags (ESTs), that have already been mapped to particularchromosomal locations. For at least some of the polynucleotide sequencesdisclosed herein, public database sequences having at least somesimilarity to the polynucleotide of the present invention have beenlisted by database accession number. Searches using the GenBankaccession numbers of these public database sequences can then beperformed at an Internet site provided by the National Center forBiotechnology Information having the addresshttp://www.ncbi.nlm.nih.gov/UniGene/, in order to identify “UniGeneclusters” of overlapping sequences. Many of the “UniGene clusters” soidentified will already have been mapped to particular chromosomalsites.

Organisms that have enhanced, reduced, or modified expression of thegene(s) corresponding to the polynucleotide sequences disclosed hereinare provided. The desired change in gene expression can be achievedthrough the use of antisense polynucleotides or ribozymes that bindand/or cleave the mRNA transcribed from the gene (Albert and Morris,1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997,Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic AcidRes. Mol. Biol. 58: 1-39; all of which are incorporated by referenceherein). Transgenic animals that have multiple copies of the gene(s)corresponding to the polynucleotide sequences disclosed herein,preferably produced by transformation of cells with genetic constructsthat are stably maintained within the transformed cells and theirprogeny, are provided. Transgenic animals that have modified geneticcontrol regions that increase or reduce gene expression levels, or thatchange temporal or spatial patterns of gene expression, are alsoprovided (see European Patent No. 0 649 464 B1, incorporated byreference herein). In addition, organisms are provided in which thegene(s) corresponding to the polynucleotide sequences disclosed hereinhave been partially or completely inactivated, through insertion ofextraneous sequences into the corresponding gene(s) or through deletionof all or part of the corresponding gene(s). Partial or complete geneinactivation can be accomplished through insertion, preferably followedby imprecise excision, of transposable elements (Plasterk, 1992,Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA90(16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2):719-722; all of which are incorporated by reference herein), or throughhomologous recombination, preferably detected by positive/negativegenetic selection strategies (Mansour et al., 1988, Nature 336: 348-352;U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396;5,616,491; and 5,679,523; all of which are incorporated by referenceherein). These organisms with altered gene expression are preferablyeukaryotes and more preferably are mammals. Such organisms are usefulfor the development of non-human models for the study of disordersinvolving the corresponding gene(s), and for the development of assaysystems for the identification of molecules that interact with theprotein product(s) of the corresponding gene(s).

Where the protein of the present invention is membrane-bound (e.g., is areceptor), the present invention also provides for soluble forms of suchprotein. In such forms, part or all of the intracellular andtransmembrane domains of the protein are deleted such that the proteinis fully secreted from the cell in which iris expressed. Theintracellular and transmembrane domains of proteins of the invention canbe identified in accordance with known techniques for determination ofsuch domains from sequence information. For example, the TopPredIIcomputer program can be used to predict the location of transmembranedomains in an amino acid sequence, domains which are described by thelocation of the center of the transmembrane domain, with at least tentransmembrane amino acids on each side of the reported centralresidue(s).

Proteins and protein fragments of the present invention include proteinswith amino acid sequence lengths that are at least 25% (more preferablyat least 50%, and most preferably at least 75%) of the length of adisclosed protein and have at least 60% sequence identity (morepreferably, at least 75% identity; most preferably at least 90% or 95%identity) with that disclosed protein, where sequence identity isdetermined by comparing the amino acid sequences of the proteins whenaligned so as to maximize overlap and identity while minimizing sequencegaps. Also included in the present invention are proteins and proteinfragments that contain a segment preferably comprising 8 or more (morepreferably 20 or more, most preferably 30 or more) contiguous aminoacids that shares at least 75% sequence identity (more preferably, atleast 85% identity; most preferably at least 95% identity) with any suchsegment of any of the disclosed proteins.

In another embodiment, proteins, protein fragments, and recombinantproteins of the present invention include those which can be identifiedbased on the presence of at least one “IL-22 receptor-binding motif.” Asused herein, the term “IL-22 receptor-binding motif” includes amino acidsequences or residues which are important for binding of IL-22 to itsrequisite receptor. In a preferred embodiment, a IL-22 protein containsa IL-22 receptor-binding motif including about amino acids 50-60 of SEQID NO:2. In another embodiment, an IL-22 protein contains a IL-22receptor-binding motif including about amino acids 63-81 of SEQ ID NO:2.In yet another embodiment, an IL-22 protein contains a IL-22receptor-binding motif including about amino acids 168-177 of SEQ IDNO:2. In a preferred embodiment, an IL-22 protein contains a IL-22receptor-binding motif including at least one of amino acids 50-60,amino acids 63-81, and/or about amino acids 168-177 of SEQ ID NO:2.

In yet another embodiment, a IL-22 receptor binding motif has an aminoacid sequence at least 95%, 96%, 97%, 98%, 99%, or more identical to anamino acid sequence selected from the group consisting of amino acids50-60 of SEQ ID NO:2, amino acids 63-81 of SEQ ID NO:2, and amino acids168-177, of SEQ ID NO:2.

In another embodiment, proteins, protein fragments, and recombinantproteins of the present invention include those which can be identifiedbased on the presence of at least one, two, three, four or more sitesfor N-linked glycosylation.

In particular, sequence identity can be determined using WU-BLAST(Washington University BLAST) version 2.0 software, which builds uponWU-BLAST version 1.4, which in turn is based on the public domainNCBI-BLAST version 1.4 (Altschul and Gish, 1996, Local alignmentstatistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschulet al., 1990, Basic local alignment search tool, Journal of MolecularBiology 215: 403-410; Gish and States, 1993, Identification of proteincoding regions by database similarity search, Nature Genetics 3:266-272; Karlin and Altschul, 1993, Applications and statistics formultiple high-scoring segments in molecular sequences, Proc. Natl. AcadSci. USA 90: 5873-5877; all of which are incorporated by referenceherein). WU-BLAST version 2.0 executable programs for several UNIXplatforms can be downloaded fromftp://blast.wustl.edu/blast/executables. The complete suite of searchprograms (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided atthat site, in addition to several support programs. WU-BLAST 2.0 iscopyrighted and may not be sold or redistributed in any form or mannerwithout the express written consent of the author, but the postedexecutables may otherwise be freely used for commercial, nonprofit, oracademic purposes. In all search programs in the suite—BLASTP, BLASTN,BLASTX, TBLASTN and TBLASTX—the gapped alignment routines are integralto the database search itself, and thus yield much better sensitivityand selectivity while producing the more easily interpreted output.Gapping can optionally be turned off in all of these programs, ifdesired. The default penalty (Q) for a gap of length one is Q=9 forproteins and BLASTP, and Q=10 for BLASTN, but can be changed to anyinteger value including zero, one through eight, nine, ten, eleven,twelve through twenty, twenty-one through fifty, fifty-one through onehundred, etc. The default per-residue penalty for extending a gap (R) isR=2 for proteins and BLASTP, and R=10 for BLASTN, but can be changed toany integer value including zero, one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve through twenty, twenty-onethrough fifty, fifty-one through one hundred, etc. Any combination ofvalues for Q and R can be used in order to align sequences so as tomaximize overlap and identity while minimizing sequence gaps. Thedefault amino acid comparison matrix is BLOSUM62, but other amino acidcomparison matrices such as PAM can be utilized.

Species homologues of the disclosed polynucleotides and proteins arealso provided by the present invention. As used herein, a “specieshomologue” is a protein or polynucleotide with a different species oforigin from that of a given protein or polynucleotide, but withsignificant sequence similarity to the given protein or polynucleotide.Preferably, polynucleotide species homologues have at least 60% sequenceidentity (more preferably, at least 75%, 80%, 85%, 90%, 95%, 99%) withthe given polynucleotide, and protein species homologues have at least30% sequence identity (more preferably, at least 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%) with the given protein, where sequenceidentity is determined by comparing the nucleotide sequences of thepolynucleotides or the amino acid sequences of the proteins when alignedso as to maximize overlap and identity while minimizing sequence gaps.Species homologues can be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source from the desired species. Preferably,species homologues are those isolated from mammalian species. Mostpreferably, species homologues are those isolated from certain mammalianspecies such as, for example, Pan troglodytes, Gorilla gorilla, Pongopygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papiohamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus,Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus,Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris,Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equuscaballus, for which genetic maps have been created allowing theidentification of syntenic relationships between the genomicorganization of genes in one species and the genomic organization of therelated genes in another species (O'Brien and Seuánez, 1988, Ann. Rev.Genet. 22: 323-351; O'Brien et al., 1993, Nature Genetics 3:103-112;Johansson et al., 1995, Genomics 25: 682-690; Lyons et al., 1997, NatureGenetics 15: 47-56; O'Brien et al., 1997, Trends in Genetics 13(10):393-399; Carver and Stubbs, 1997, Genome Research 7:1123-1137; all ofwhich are incorporated by reference herein)

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotides which also encode proteins whichare identical or have significantly similar sequences to those encodedby the disclosed polynucleotides. Preferably, allelic variants have atleast 60% sequence identity (more preferably, at least 75%, 80%, 85%,90%, 95%, 99%) with the given polynucleotide, where sequence identity isdetermined by comparing the nucleotide sequences of the polynucleotideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. Allelic variants can be isolated and identified by makingsuitable probes or primers from the sequences provided herein andscreening a suitable nucleic acid source from individuals of theappropriate species.

The invention also includes polynucleotides with sequences complementaryto those of the polynucleotides disclosed herein.

The present invention also includes polynucleotides that hybridize underreduced stringency conditions, more preferably stringent conditions, andmost preferably highly stringent conditions, to polynucleotidesdescribed herein. Examples of stringency conditions are shown in thetable below: highly stringent conditions are those that are at least asstringent as, for example, conditions A-F; stringent conditions are atleast as stringent as, for example, conditions G-L; and reducedstringency conditions are at least as stringent as, for example,conditions M-R.

Poly- Stringency nucleotide Hybrid Hybridization Temperature and WashTemperature Condition Hybrid Length (bp)^(‡) Buffer^(†) and Buffer^(†) ADNA:DNA ≧50 65° C.; 1xSSC -or- 65° C.; 0.3xSSC 42° C.; 1xSSC, 50%formamide B DNA:DNA <50 T_(B)*; 1xSSC T_(B)*; 1xSSC C DNA:RNA ≧50 67°C.; 1xSSC -or- 67° C.; 0.3xSSC 45° C.; 1xSSC, 50% formamide D DNA:RNA<50 T_(D)*; 1xSSC T_(D)*; 1xSSC E RNA:RNA ≧50 70° C.; 1xSSC -or- 70° C.;0.3xSSC 50° C.; 1xSSC, 50% formamide F RNA:RNA <50 T_(F)*; 1xSSC T_(F)*;1xSSC G DNA:DNA ≧50 65° C.; 4xSSC -or- 65° C.; 1xSSC 42° C.; 4xSSC, 50%formamide H DNA:DNA <50 T_(H)*; 4xSSC T_(H)*; 4xSSC I DNA:RNA ≧50 67°C.; 4xSSC -or- 67° C.; 1xSSC 45° C.; 4xSSC, 50% formamide J DNA:RNA <50T_(J)*; 4xSSC T_(J)*; 4xSSC K RNA:RNA ≧50 70° C.; 4xSSC -or- 67° C.;1xSSC 50° C.; 4xSSC, 50% formamide L RNA:RNA <50 T_(L)*; 2xSSC T_(L)*;2xSSC M DNA:DNA ≧50 50° C.; 4xSSC -or- 50° C.; 2xSSC 40° C.; 6xSSC, 50%formamide N DNA:DNA <50 T_(N)*; 6xSSC T_(N)*; 6xSSC O DNA:RNA ≧50 55°C.; 4xSSC -or- 55° C.; 2xSSC 42° C.; 6xSSC, 50% formamide P DNA:RNA <50T_(P)*; 6xSSC T_(P)*; 6xSSC Q RNA:RNA ≧50 60° C.; 4xSSC -or- 60° C.;2xSSC 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 T_(R)*; 4xSSC T_(R)*;4xSSC ^(‡)The hybrid length is that anticipated for the hybridizedregion(s) of the hybridizing polynucleotides. When hybridizing apolynucleotide to a target polynucleotide of unknown sequence, thehybrid length is assumed to be that of the hybridizing polynucleotide.When polynucleotides of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal sequence complementarity.^(†)SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4)can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodiumcitrate) in the hybridization and wash buffers; washes are performed for15 minutes after hybridization is complete. *T_(B)-T_(R): Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m)(° C.) = 2(# of A + T bases) + 4(# of G + C bases). Forhybrids between 18 and 49 base pairs in length, T_(m)(° C.) = 81.5 +16.6(log₁₀[Na⁺]) + 0.41(% G + C) − (600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1xSSC = 0.165M).

Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, P. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference.

Preferably, each such hybridizing polynucleotide has a length that is atleast 25% (more preferably at least 50%, and most preferably at least75%) of the length of the polynucleotide of the present invention towhich it hybridizes, and has at least 60% sequence identity (morepreferably, at least 75% identity; most preferably at least 90% or 95%identity) with the polynucleotide of the present invention to which ithybridizes, where sequence identity is determined by comparing thesequences of the hybridizing polynucleotides when aligned so as tomaximize overlap and identity while minimizing sequence gaps.

II. Vectors and Host Cells

The isolated polynucleotide of the invention can be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids. Res. 19, 4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it can be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itcan be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments can beaccomplished using known chemical or enzymatic methods. The protein mayalso be modified by covalent modifications including, but not limited,polyethylene glycol modifications.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

The protein of the invention can be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it can beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLabs (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and Invitrogen Corporation (Carlsbad, Calif.),respectively. The protein can also be tagged with an epitope andsubsequently purified by using a specific antibody directed to suchepitope. One such epitope (“Flag”) is commercially available from theEastman Kodak Company (New Haven, Conn.).

In addition, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g.; silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. Thus, they can be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The protein may also be produced in a recombinant viral vector throughtechniques that are well-known in the art. For example, recombinantadenoviruses, such as the Ad5 E1a deleted (dl327) recombinantadenovirus, can be generated through homologous recombination in a humankidney embryonic cell line. IL-22 cDNA can then be ligated into anadenovirus vector such as Adori 1-2. The cloned viral vector can then beadministered to a subject via subcutaneous or intravenous injection toallow for in vivo production of the invention.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications in the peptide or DNA sequences can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues can be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and may thusbe useful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are believed to be encompassed by the presentinvention.

III. Methods of Use and Biological Activity

The polynucleotides and proteins of the present invention can exhibitone or more of the biological activities (including those associatedwith assays cited herein) identified below and accordingly are useful ina variety of research, pharmaceutical and therapeutic methods. Methods,uses or activities described for proteins of the present invention canbe provided by administration or use of such proteins or byadministration or use of polynucleotides encoding such proteins (suchas, for example, in gene therapies or vectors suitable for introductionof DNA).

The structural and functional properties of IL-22 place this protein inthe cytokine family. Cytokines play important roles both in health anddisease and have multiple clinical indications. As is described indetail in the Examples, below, IL-22 induces changes associated withthose caused by inflammatory cytokines (such as IL-1 and TNFα), andinhibitors of IL-22 ameliorate symptoms of rheumatoid arthritis,Therefore, IL-22, and/or agents that increase levels of IL-22 or mimicthe actions of IL-22 (and other molecules of the present invention) areuseful as agonists in certain clinical indications, and antagonists ofthis molecule are useful in other clinical situations, particularly inthose in which modulation of an inflammatory state is desired. Whetherthe agonist or antagonist is the preferred depends on the particularaspects of the disease pathology, such as the cell types involved, thenature of the stimulus and the cellular microenvironment.

In a preferred embodiment, IL-22 activity includes induction of at leastone activity indicative of an inflammatory state. Additional activitiescan include at least one or more of the following activities: (1)modulating, for example antagonizing a signal transduction pathway (e.g.an IL-22 dependant pathway); (2) modulating cytokine production and/orsecretion (e.g. production and/or secretion of a proinflammatorycytokine); (3) modulating lymphokine production and/or secretion; (4)modulating production of adhesion molecules and/or cellular adhesion;(5) modulating expression or activity of nuclear transcription factors;(7) modulating secretion of IL-1; (8) competing with receptors for othercytokines; (9) competing with another IL-22 family member protein tobind a IL-22 receptor; (10) modulating nuclear translocation ofinternalized receptor for IL-22 or another cytokine or ligand-complexedreceptor; (11) modulating cell proliferation, development ordifferentiation, for example, cytokine-stimulated or a IL-22protein-stimulated proliferation, development or differentiation (e.g.,of an epithelial cell, for example, a squamous epithelial cell of theesophagus, or of a skin cell, e.g., a keratinocyte); (12) modulatingcell proliferation, development or differentiation of an osteogenic cell(e.g., of an osteoclast precursor cell, osteoclast and/or osteoblast);(13) modulating bone formation, bone metabolism and/or bone homeostasis(e.g., inhibiting bone resorption); (15) modulating cellular immuneresponses; (16) modulating cytokine-mediated proinflammatory actions(e.g., inhibiting acute phase protein synthesis by hepatocytes, fever,and/or prostaglandin synthesis, for example PGE₂ synthesis); and (17)promoting and/or potentiating wound healing.

Examples of IL-22 inhibitors include soluble fragments of IL-22polypeptides. The soluble fragments can be provided as fusion proteins,e.g., as IgG fusion proteins. Inhibitors can additionally includeantibodies to IL-22 polypeptides, as well as small molecule inhibitorsof IL-22 polypeptides. The small molecules can act by inhibiting theexpression and/or activity of an IL-22 polypeptide.

A. Diagnostic Assays

An exemplary method for detecting the presence or absence of IL-22protein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting IL-22 protein ornucleic acid (e.g., mRNA, genomic DNA) that encodes IL-22 protein suchthat the presence of IL-22 protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting IL-22 mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to IL-22 mRNAor genomic DNA. The nucleic acid probe can be, for example, afull-length IL-22 nucleic acid, such as the nucleic acid of SEQ ID NO:1, or a fragment or portion of an IL-22 nucleic acid such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to IL-22 mRNA or genomic DNA. Other suitable probes for usein the diagnostic assays of the invention are described herein.

A preferred agent for detecting IL-22 protein is an antibody capable ofbinding to IL-22 protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect IL-22 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of IL-22 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of IL-22 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of IL-22 genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of IL-22 protein include introducing into a subject a labeledanti-IL-22 antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting IL-22 protein, mRNA, orgenomic DNA, such that the presence of IL-22 protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofIL-22 protein, mRNA or genomic DNA in the control sample with thepresence of IL-22 protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of IL-22in a biological sample. For example, the kit can comprise a labeledcompound or agent (e.g. probe or antibody) capable of detecting IL-22protein or mRNA in a biological sample; means for determining the amountof IL-22 in the sample; and means for comparing the amount of IL-22 inthe sample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect IL-22 protein or nucleic acid.

Human IL-22 agonists include without limitation human IL-22 proteins andfragments, deletion mutants and addition mutants thereof; and peptideand small molecule compounds that interact with the receptor or othertarget to which human IL-22 is directed. Human IL-22 antagonists includewithout limitation antibodies directed to human IL-22 proteins; solubleforms of the receptor or other target to which human IL-22 is directed;antibodies directed to the receptor or other target to which human IL-22is directed; and peptide and small molecule compounds that inhibit orinterfere with the interaction of human IL-22 with its receptor or othertarget.

B. Pharmaceutical Compositions

The nucleic acid molecules, proteins, modulatory agents, and/orantibodies and biosynthetic molecules (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein,modulatory agents, and/or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions. Thepharmaceutical composition of the invention may also contain additionalcytokines, lymphokines, or other hematopoietic factors. Thepharmaceutical composition may further contain other agents which eitherenhance the activity of the protein or compliment its activity or use intreatment.

Thus, the pharmaceutical composition of the invention may also containadditional cytokines, lymphokines, or other hematopoietic factors suchas M-CSF, G-CSF, GM-CSF, Meg-GCSF, thrombopoietin, stem cell factor,erythropoietin, TNFα, IL-1β, IL-2 through IL-26, IFNα/b, IFNγ, as wellas inhibitors of all of the above cytokines, particularly inhibitors ofTNFα, IL-1β, IL-12 and IL-18.

Such additional factors and/or agents can be included in thepharmaceutical composition to produce a synergistic effect with proteinof the invention, or to minimize side effects. Conversely, protein ofthe present invention can be included in formulations of the particularcytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent to minimize sideeffects of the cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

A protein of the present invention can be active in multimers (e.g.,heterodimers or homodimers) or complexes with itself or other proteins.As a result, pharmaceutical compositions of the invention may comprise aprotein of the invention in such multimeric or complexed form.

The pharmaceutical composition of the invention can be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunolgobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention can be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No.4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all ofwhich are incorporated herein by reference.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such is mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a Immunomodulin protein or anti-Immunomodulin antibody)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile, salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, a “Therapeutically effective” dose canbe estimated initially from cell culture assays. A “therapeuticallyeffective” dose can be further formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system. ex vivo

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

C. Therapeutic Uses

The present invention provides for both prophylactic and therapeuticmethods of treating subjects (e.g., human subjects). In one aspect, theinvention provides a method for preventing or treating a disease or adisorder in a subject prophylactically or therapeutically.Administration of an agent prophylactically can occur prior to themanifestation of symptoms of an undesired disease or disorder, such thatthe disease or disorder is prevented or, alternatively, delayed in itsprogression. The prophylactic methods of the present invention can becarried out in a similar manner to therapeutic methods described herein,although dosage and treatment regimes may differ.

Another aspect of the invention pertains to methods for treating asubject therapeutically. In one embodiment, the present inventionincludes methods of modulating an immune response. In particular,modulation of an immune response includes, but is not limited to,modulation of cellular toxicity, modulation of cytokine expression,production or secretion (e.g., enhancement or inhibition of cytokineexpression, production or secretion). A preferred embodiment of theinvention involves modulation of IL-22, in particular, stimulation ofIL-22 using a IL-22 stimulatory modulator or, alternatively, inhibitionof IL-22 using a IL-22 inhibitory modulator. Accordingly, the presentmethod has therapeutic utility in biasing an immune response towards, oraway from, a natural immunity-type response depending upon the desiredtherapeutic regimen. Such modulatory methods are particularly useful indiseases such as viral and bacterial infection, in particular acutephase responses, sepsis and autoimmune disorders (i.e. chronicinflammatory states). Moreover, the immunomodulatory methods of thepresent invention can be used to treat an immunocompromised individualto enhance immunity. Uses to increase resistance to viral infection andenhance the rejection of foreign molecules are also within the scope ofthe present invention. The immunomodulatory methods of the presentinvention are further useful in treating sepsis. For example, aninhibition of cytokines such as IL-22 and Tumor Necrosis Factor inpatients infected with gram negative bacteria can result in anattenuated immune response, thus preventing septic shock. Theimmunomodulatory methods of the present invention are further useful intreating acute phase responses and chronic inflammatory diseases.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

As used herein, the term “treatment” includes the application oradministration of a therapeutic agent to a subject or to an isolatedtissue or cell line from a subject, who is afflicted with a disease, asymptom of disease or a predisposition toward a disease, with the goalof curing, healing, alleviating, relieving, altering, remedying,ameliorating, improving or affecting the disease, the symptoms ofdisease or the predisposition toward disease.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention can be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention can be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

Administration of protein of the present invention used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral or intravenous injection.Intravenous administration to the patient is preferred.

When a therapeutically effective amount of protein of the presentinvention is administered orally, protein of the present invention willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%protein of the present invention, and preferably from about 25 to 90%protein of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils can be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein of the present invention, and preferably from about 1to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention can be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 ng to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the protein of the present inventionwill be in the range of 12 to 24 hours of continuous intravenousadministration. Ultimately the attending physician will decide on theappropriate duration of intravenous therapy using the pharmaceuticalcomposition of the present invention.

In a preferred embodiment, pharmaceutical preparations (e.g. thosecomprising neutralizing agents against) of the present invention will beadministered 2 to 6 hours after onset of infection. For example, invitro assays suggest that adherent compartment of peritoneal exudatecells produce IL-22 2 to 6 hours after treatment with LPS, suggestingthat IL-22 is produced early in an immune response. Accordingly, earlyadministration of, for example, neutralizing agents, during the courseof infection may enhance the therapeutic efficacy of such agents.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies which specifically react with theprotein. As used herein, the term “antibody” includes without limitationa polyclonal antibody, a monoclonal antibody, a chimeric antibody, asingle-chain antibody, a CDR-grafted antibody, a humanized antibody, orfragments thereof which bind to the indicated protein. Such term alsoincludes any other species derived from an antibody or antibody sequencewhich is capable of binding the indicated protein.

Antibodies to a particular protein can be produced by methods well knownto those skilled in the art. For example, monoclonal antibodies can beproduced by generation of antibody-producing hybridomas in accordancewith known methods (see for example, Goding, 1983, Monoclonalantibodies: principles and practice, Academic Press Inc, New York; andYokoyama, 1992, “Production of Monoclonal Antibodies” in CurrentProtocols in Immunology, Unit 2.5, Greene Publishing Assoc. and JohnWiley & Sons). Polyclonal sera and antibodies can be produced byinoculation of a mammalian subject with the relevant protein orfragments thereof in accordance with known methods. Examples of suitablemethods include direct DNA gene or viral (e.g., adenovirus orretroviral) administration to an animal. Fragments of antibodies,receptors, or other reactive peptides can be produced from thecorresponding antibodies by cleavage of and collection of the desiredfragments in accordance with known methods (see for example, Goding,supra; and Andrew et al., 1992, “Fragmentation of Immunoglobulins” inCurrent Protocols in Immunology, Unit 2.8, Greene Publishing Assoc. andJohn Wiley & Sons). Chimeric antibodies and single chain antibodies canalso be produced in accordance with known recombinant methods (see forexample, U.S. Pat. Nos. 5,169,939, 5,194,594, and 5,576,184). Humanizedantibodies can also be made from corresponding murine antibodies inaccordance with well known methods (see for example, U.S. Pat. Nos.5,530,101, 5,585,089, and 5,693,762). Additionally, human antibodies canbe produced in non-human animals such as mice that have been geneticallyaltered to express human antibody molecules (see for example Fishwild etal., 1996, Nature Biotechnology 14: 845-851; Mendez a al., 1997, NatureGenetics 15: 146-156 (erratum Nature Genetics 16: 410); and U.S. Pat.Nos. 5,877,397 and 5,625,126). Such antibodies can be obtained usingeither the entire protein or fragments thereof as an immunogen. Thepeptide immunogens additionally may contain a cysteine residue at thecarboxyl terminus, and are conjugated to a hapten such as keyhole limpethemocyanin (KLH). Methods for synthesizing such peptides are known inthe art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85,2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

Monoclonal antibodies binding to the protein of the invention is usefulas diagnostic agents for the immunodetection of the protein.Neutralizing monoclonal antibodies binding to the protein may also beuseful therapeutics for both conditions associated with the protein andalso in the treatment of some forms of cancer where abnormal expressionof the protein is involved. In the case of cancerous cells or leukemiccells, neutralizing monoclonal antibodies against the protein are usefulin detecting and preventing the metastatic spread of the cancerouscells, which can be mediated by the protein.

In a preferred embodiment, monoclonal and polyclonal antibodies are usedto down-modulate an immune response. Examples of immunologicalconditions which can benefit from such treatment include bacterialinfections (i.e. induction of sepsis that can lead to septic shockand/or septicemia) and other chronic inflammatory conditions such asrheumatoid arthritis and osteoarthritis.

Agents which modulate the activity of the present invention are alsoused to alter the inflammatory pathologies of the kidney.

For compositions of the present invention which arc useful for bone,cartilage, tendon or ligament regeneration, the therapeutic methodincludes administering the composition topically, systematically, orlocally as an implant or device. When administered, the therapeuticcomposition for use in this invention is, of course, in a pyrogen-free,physiologically acceptable form. Further, the composition may desirablybe encapsulated or injected in a viscous form for delivery to the siteof bone, cartilage or tissue damage. Topical administration can besuitable for wound healing and tissue repair. Therapeutically usefulagents other than a protein of the invention which may also optionallybe included in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing composition to the site of bone and/orcartilage damage, providing a structure for the developing bone andcartilage and optimally capable of being resorbed into the body. Suchmatrices can be formed of materials presently in use for other implantedmedical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions can be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sintered hydroxapatite,bioglass, aluminates, or other ceramics. Matrices can be comprised ofcombinations of any of the above mentioned types of material, such aspolylactic acid and hydroxyapatite or collagen and tricalciumphosphate.The bioceramics can be altered in composition, such as incalcium-aluminate-phosphate and processing to alter pore size, particlesize, particle shape, and biodegradability.

Presently preferred is a 50:50 (mole weight) copolymer of lactic acidand glycolic acid in the form of porous particles having diametersranging from 150 to 800 microns. In some applications, it will be usefulto utilize a sequestering agent, such as carboxymethyl cellulose orautologous blood clot, to prevent the protein compositions fromdisassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells.

In further compositions, proteins of the invention can be combined withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in question. These agents include variousgrowth factors such as epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins of the present invention.

The dosage regimen of a protein-containing pharmaceutical composition tobe used in tissue regeneration will be determined by the attendingphysician considering various factors which modify the action of theproteins, e.g., amount of tissue weight desired to be formed, the siteof damage, the condition of the damaged tissue, the size of a wound,type of damaged tissue (e.g., bone), the patient's age, sex, and diet,the severity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA).

Cells may also be cultured ex vivo in the presence of proteins of thepresent invention in order to proliferate or to produce a desired effecton or activity in such cells. Treated cells can then be introduced invivo for therapeutic purposes.

D. Screening Assays

The proteins provided by the present invention can be used in assays todetermine biological activity, including in a panel of multiple proteinsfor high-throughput screening; to raise antibodies or to elicit anotherimmune response; as a reagent (including the labeled reagent) in assaysdesigned to quantitatively determine levels of the protein (or itsreceptor) in biological fluids; as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state); and, of course, to isolate correlative receptors orligands. Where the protein binds or potentially binds to another protein(such as, for example, in a receptor-ligand interaction), the proteincan be used to identify the other protein with which binding occurs orto identify inhibitors of the binding interaction. Proteins involved inthese binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, WStrober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immuno).137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins, that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wileyand Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965,1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

Modulatory agents identified by the above-described screening assays aretested in an appropriate animal model, for example, to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, modulatory agents are tested in at least one of the invitro or in situ assays described herein.

In another aspect of the invention, transgenic and gene knockout animalsare used to study disease. Specifically, mice that have been geneticallyaltered to express a disease phenotype are used to screen agents thatmodulate IL-22 activity. As used herein, the term “genetically altered”means any animal that has manipulated genetically, either by theintroduction of a heterologous gene encoding a protein (a transgenicanimal) or by the deletion of a gene by homologous recombination (a geneknockout animal). Preferably, the animal that has been geneticallyaltered is a mouse.

In another embodiment, IL-22 molecules (e.g. RNA, DNA, cDNA, protein orantibodies) are used as diagnostic tools, e.g., to detect the presenceof tissues in an inflammatory state.

Assaying Effects of IL-22 Modulator

The activity of an IL-22 agonist or antagonist can be measure by thefollowing methods:

Assays for T-cell or thymocyte proliferation include without limitationthose, described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeck, D. H. Margulies, E. M. Shevach, W Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. Coligan eds.Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human Interferon γ, Schreiber, R. D. In CurrentProtocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, JohnWiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E.e.a. Coligan eds. Vol 1 pp. 63.1-6.3.12, John Wiley andSons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991;Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl.Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and humaninterleukin 6—Nordan, R. In Current Protocols in Immunology. J. E.e.a.Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E.e.a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger ct al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

Immune Stimulating or Suppressing Activity

An IL-22 modulator may also exhibit immune stimulating or immunesuppressing activity, including without limitation the activities forwhich assays are described herein. An IL-22 agonist is useful in thetreatment of various immune deficiencies and disorders (including severecombined immunodeficiency (SCID)), e.g., in regulating (up or down)growth and proliferation of T and/or B lymphocytes, as well as effectingthe cytolytic activity of NK cells and other cell populations. Theseimmune deficiencies can be genetic or be caused by viral (e.g., HIV) aswell as bacterial or fungal infections, or may result from autoimmunedisorders. More specifically, infectious diseases causes by viral,bacterial, fungal or other infection can be treatable using a protein ofthe present invention, including infections by HIV, hepatitis viruses,herpesviruses, mycobacteria, Leishmania spp, malaria spp. and variousfungal infections such as candidiasis. An IL-22 protein is also usefulwhere a boost to the immune system generally can be desirable, i.e., inthe treatment of cancer.

An IL-22 inhibitor (such as an IL-22 antibody) can be used to treat anautoimmune disorder. Autoimmune disorders that can be treated using aprotein of the present invention include, for example, connective tissuedisease, multiple sclerosis, systemic lupus erythematosus, rheumatoidarthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome,autoimmune thyroiditis, insulin dependent diabetes mellitis, myastheniagravis, graft-versus-host disease and autoimmune inflammatory eyedisease. Such a protein of the present invention can also be used totreat inflammatory conditions associated with, e.g., pancreatitis. IL-22inhibitors are also useful in the treatment of allergic reactions andconditions, such as asthma (particularly allergic asthma) or otherrespiratory problems. Other conditions, in which immune suppression isdesired (including, for example, organ transplantation), may also betreatable using a protein of the present invention.

Using the proteins of the invention it may also be possible to regulateimmune responses in a number of ways. Down regulation can be in the formof inhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells can be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

As used herein, the term “pathological condition” refers to thestructural and functional consequences of injurious stimuli on cells,tissues, and organs and ultimately the consequences on the entireorganism. Such injurious stimuli includes, but is not limited to,infection with a foreign body (i.e. bacteria, virus, fungi andparasite), inflammation, autoimmune disorders (i.e. rheumatoidarthritis, osteoarthritis, multiple sclerosis, myasthenia gravis,inflammatory bowel diseases, diabetes, SLE, and psoriasis), cancer,necrosis, ischemia, acute phase responses, apoptosis, wound healingprocesses, cholesterol metabolism, oxygen free radical injury,atherosclerosis and allergies.

In a particular embodiment, the down-regulation or preventing of one ormore activities of the IL-22 molecules of present invention can behelpful in the treatment of sepsis. Briefly, sepsis is an out of controlinflammatory response, often in response to various pus-forming andother pathogenic organisms (i.e. gram negative bacteria), or theirtoxins, in the blood or tissues. Agents that block the activity of IL-22molecules of the present invention can, accordingly, attenuate theinduction of septic shock.

In another embodiment, down regulating or preventing one or more antigenfunctions (including without limitation B lymphocyte antigen functions(such as, for example, B7)), e.g., preventing high level lymphokinesynthesis by activated T cells, will be useful in situations of tissue,skin and organ transplantation and in graft-versus-host disease (GVHD).For example, blockage of T cell function should result in reduced tissuedestruction in tissue transplantation. Typically, in tissue transplants,rejection of the transplant is initiated through its recognition asforeign by T cells, followed by an immune reaction that destroys thetransplant. The administration of a molecule which inhibits or blocksinteraction of a B7 lymphocyte antigen with its natural ligand(s) onimmune cells (such as a soluble, monomeric form of a peptide having B7-2activity alone or in conjunction with a monomeric form of a peptidehaving an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) orblocking antibody), prior to transplantation can lead to the binding ofthe molecule to the natural ligand(s) on the immune cells withouttransmitting the corresponding costimulatory signal. Blocking Blymphocyte antigen function in this matter prevents cytokine synthesisby immune cells, such as T cells, and thus acts as an immunosuppressant.Moreover, the lack of costimulation may also be sufficient to anergizethe T cells, thereby inducing tolerance in a subject. Induction oflong-term tolerance by B lymphocyte antigen-blocking reagents may avoidthe necessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA 41 g fusionproteins in vivo as described in Lenschow et al., Science 257:789-792(1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105(1992). In addition, murine models of GVHD (see Paul ed., FundamentalImmunology, Raven Press, New York, 1989, pp. 846-847) can be used todetermine the effect of blocking B lymphocyte antigen function in vivoon the development of that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which can be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840.856).

In a particular embodiment, IL-22 inhibitory agents (e.g. blockingagents) of the present invention can be used to treat autoimmunedisease, in particular, lymphocyte-mediated autoimmune diseases,associated with prolonged acute phase responses that persist in thesetting of chronic inflammation, such as rheumatoid arthritis,osteoarthritis, multiple sclerosis, inflammatory bowel disease, diabetesand Systemic Lupus Erythomatosis (SLE), atherosclerosis and allergies.In such a situation, persistent elevations of serum amyloid A proteinmay lead to deposition of this protein in the interstitium of tissues, acondition known as amyloidosis. The deposition of serum amyloid A intissues is often in the form of fibrils rich in β-pleated sheetstructures which can interfere with normal tissue function (i.e.myocardial contraction, glomerular filtration). Thus, inhibiting orblocking IL-22 production, for example, by a neutralizing antibody canhelp stop the acute phase reaction, and therefore prevent the occurrenceof amyloidosis.

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses can be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function is useful in cases of viral infection. Inaddition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses can be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

In a particular embodiment, the IL-22 molecules and/or modulators of thepresent invention can be used as a vaccine adjuvant due to their abilityto modulate the immune system. For example, the IL-22 molecules and/ormodulators (e.g. positive modulatory agents) of the present inventioncan be co-administered with a potential vaccine antigen in order toelicit a nonspecific inflammatory immune response to the potentialvaccine antigen. Such vaccines can be directed against a foreignorganism (i.e. bacteria, virus, parasite or fungi) or a tumor antigen.Moreover, such treatments may include, but are not limited to, genetransfer with IL-22 DNA.

As used herein, the term “immunogenicity-augmenting” includes enhancingand/or increasing the immunogenicity of, for example, a vaccine, ascompared to such vaccine in the absence of IL-22.

In another application, up regulation or enhancement of antigen function(preferably B lymphocyte antigen function) is useful in the induction oftumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma,leukemia, neuroblastoma, carcinoma) transfected with a nucleic acidencoding at least one peptide of the present invention can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides. For example, tumor cells obtained from apatient can be transfected ex vivo with an expression vector directingthe expression of a peptide having B7-2-like activity alone, or inconjunction with a peptide having B7-1-like activity and/or B7-3-likeactivity. The transfected tumor cells are returned to the patient toresult in expression of the peptides on the surface of the transfectedcell. Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activityof a B lymphocyte antigen(s) on the surface of the tumor cell providesthe necessary costimulation signal to T cells to induce a T cellmediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient amounts of MHC class I or MHCclass II molecules, can be transfected with nucleic acid encoding all ora portion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I a chain protein and β₂ microglobulin protein or an MHC class IIαchain protein and an MHC class II β chain protein to thereby express MHCclass I or MHC class II proteins on the cell surface. Expression of theappropriate class I or class II MHC in conjunction with a peptide havingthe activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) inducesa T cell mediated immune response against the transfected tumor cell.Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject can besufficient to overcome tumor-specific tolerance in the subject.

Hematopoiesis Regulating Activity

An IL-22 protein, or modulator can also be used to regulatehematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, In Culture of Hematopoietic Cells. R. I. Freshney, et al.eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bonemarrow cultures in the presence of stromal cells, Spooncer, E., Dexter,M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Longterm culture initiating cell assay, Sutherland, H. J. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162,Wiley-Liss, Inc., New York, N.Y. 1994.

Tissue Growth Activity

A protein of the present invention also may have utility in compositionsused for bone, cartilage, tendon, ligament and/or nerve tissue growth orregeneration, as well as for wound healing and tissue repair andreplacement, and in the treatment of burns, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein of the invention may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes.

Another category of tissue regeneration activity that can beattributable to the protein of the present invention is tendon/ligamentformation. A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide an environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendonitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein can be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which can betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like.

A protein of the present invention also exhibits activity for generationor regeneration of other tissues, such as organs (including, forexample, pancreas, liver, intestine, kidney, skin, endothelium), muscle(smooth, skeletal or cardiac) and vascular (including vascularendothelium) tissue, or for promoting the growth of cells comprisingsuch tissues. Part of the desired effects can be by inhibition ormodulation of fibrotic scarring to allow normal tissue to regenerate. Aprotein of the invention may also exhibit angiogenic activity.

A protein of the present invention may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage.

In a preferred embodiment, the present invention is used for there-modeling of kidney tissue, both ex vivo and in vivo. For example,exogenous IL-22 induces the generation of epithelial tissue in theproximal tubules of the kidney.

A protein of the present invention is also useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

The activity of a protein of the invention can be measured by methodsknown in the art. Some of these are described below:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H Iand Royce, D T, eds.), Year Book Medical Publishers, Inc., Chicago, asmodified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolyticactivity. As a result, such a protein is expected to be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A protein of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Chin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79(1991); Schaub, Prostaglandins35:467-474, 1988.

Anti-Inflammatory Activity

IL-22 antagonists can be used as anti-inflammatory agents. Suitableconditions (including chronic or acute conditions), including withoutlimitation inflammation associated with infection (such as septic shock,sepsis or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine-induced lung injury, inflammatory bowel disease, Crohn'sdisease or resulting from over production of cytokines such as TNF orIL-1. Additional indications include anaphylaxis and hypersensitivity toan antigenic substance or material.

This invention is further illustrated by the non-limiting examples. Thecontents of all references, patents and published patent applicationscited throughout this application, as well as the Sequence Listing, areincorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Clone “IL-22”

A polynucleotide of the present invention has been identified as clone“IL-22”. Clone IL-22 was isolated according to the following method. Amurine EST was identified from a murine cDNA library made fromsplenocytes activated with both ConA and bone marrow derived dendriticcells. The EST was identified using methods which are selective forcDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637). Themurine EST sequence was used to isolate a full-length murine clone fromthe same cDNA library. Analysis of the sequence of the murine clonerevealed a significant homology to interleukin-10 (IL-10).

In order to isolate a human homolog of the murine clone, PCR primerswere constructed based upon the region of the murine sequence whichshowed homology to IL-10. Use of such primers for amplification in acDNA library derived from PHA/PMA-stimulated human PBMCs produced a PCRproduct of significant size. Analysis of the sequence of the PCR productconfirmed that it was a homolog of the murine cDNA. Oligonucleotideswere constructed from the sequence of the partial human clone and usedto isolate a full-length human clone from the PBMC library.

IL-22 is a full-length human clone, including the entire coding sequenceof a secreted protein (also referred to herein as “IL-22” protein).Analysis of its amino acid sequence indicated that it has about 23%homology to hIL-10. Based on the putative receptor-binding motifs inIL-10, three motifs involved with analogous function have been proposedin IL-22 through computer modeling. These are the regions of SEQ ID NO:2from residue 50 to 60, from residue 63 to 81, and from residue 168 to177. Analyses of databases revealed that IL-22 also exhibits similarlevels of homology with IL-10 of other species.

The nucleotide sequence of IL-22 as presently determined is reported inSEQ ID NO:1, and includes a poly(A) tail. The amino acid sequence of theIL-22 protein corresponding to the foregoing nucleotide sequence isreported in SEQ ID NO:2.

Example 2 Characterization of IL-22 Protein

Cell lines which stably express and secrete full length IL-22 proteinwere created by transfecting CHO cells with IL-22 cDNA in appropriateexpression vectors. Transiently transfected COS cells using appropriateIL-22 expression vectors have been used to make IL-22 protein foranalysis. Transfections were accomplished using the commerciallyavailable Lipofectamine reagent (Gibco). Interestingly, COS cells whichexpress IL-22 were observed to non-uniformly detach, forming holes inthe cell culture monolayer. Media conditioned by transfected COS cellswas used to demonstrate cytokine-like activity of IL-22 protein. Westernblot analysis of cell lysates showed that Stat-3 becomes phosphorylated(activated) in a kidney mesangial tissue-derived cell line exhibitingmacrophage-like qualities (MES-13; see, Dumoutier et al (2000) J. ofImmunology 164:1814-1819) upon exposure of that cell to mediaconditioned by IL-22-expressing cells. In addition phosphorylation ofStat-3 is induced in non-transfected COS cells that are treated withIL-22 protein.

Electrophoretic analysis of IL-22 protein (derived from the transfectedCOS cell lines described herein) indicated that the expressed proteinexists in a range of sizes. Treatment of COS-derived IL-22 protein withN-glycanase prior to electrophoresis results in a single bandcorresponding to the highest mobility (e.g. lowest molecular weight)species seen in untreated IL-22. This is consistent with proposedglycosylation events which may occur at the putative N-linkedglycosylation sites identified in the amino acid sequence of IL-22(amino acid residues 54-56, 68-70, 97-99, and 176-178 of SEQ ID NO:2).

Edman N-terminal sequencing determined that the N-terminus of the matureIL-22 protein begins with the residue at position 34 of SEQ ID NO:2(alanine). Expression vectors were created which fuse a “6× histidine”affinity tag and a FLAG epitope tag to the N-terminus of the matureIL-22 protein. (The added amino acid tag is given in SEQ ID NO:5 and hasthe following amino acid sequence:MKFLVNVALVFMVVYISYIYAGSGHHHHHHGSGDYKDDDDKAPISSHCR). These taggedconstructs were used to create stably expressing CHO cell lines andtransiently expressing COS cell lines. The tags provided a convenientmeans for detecting IL-22 (e.g., anti-6× his antibodies; anti-FLAGantibodies), and for purifying the protein from conditioned media (usingNi³⁰ ² resin). Human IL-22 protein purified by this tag from theIL-22-expressing COS cell lines could used to induce Stat-3 activationin MES-13 cells.

Comparison of IL-22 mRNA transcripts in activated Th1 and Th2 cells(see, for example, Syrbe et al, (1999) Springer Seminars inImmunopathology, 21:263-85) indicated a substantially higher level ofexpression of IL-22 in activated Th1 cells than inactivated Th2 cells.Analysis of IL-22 mRNA was accomplished with RNAse protection assays.Therefore, IL-22 is induced during an adaptive immune response,specifically by Th1 CD4+ T cells.

Example 3 Establishment of IL-22 Recombinant Adenovirus Vector and inVivo Administration

The Adori 1-2 murine IL-22 (mIL-22) vector was derived by digestingpED6dpc-2mIL-22 with EcoRI and NotI, and ligating the 1.1 kb mIL-22 cDNAfragment with EcoRI and NotI digested adenovirus vector Adori 1-2. Adori1-1 green fluorescent protein (GFP) construct was derived by digestingpEGFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, Calif.) with EcoRI andNotI and inserting the EGFP into the EcoRI and NotI site of Adori 1-1.Both constructs were verified by extensive restriction digestionanalysis and sequencing of the cDNA inserts within the plasmids.Expression of the mIL-22 cDNA and EGFP are driven from cytomegalovirus(CMV) immediate early promoter and enhancer.

Ad5 E1a deleted (dl327) recombinant adenovirus was generated byhomologous recombination in a human kidney embryonic kidney cell line293. Recombinant adenovirus virus was isolated and subsequentlyamplified on 293 cells. The virus was released from infected 293 cellsby three cycles of freeze thawing. The virus was further purified by twocesium chloride centrifugation gradients and dialyzed against phosphatebuffered saline (PBS) pH 7.2 at 4° C. Following dialysis, glycerol wasadded to a concentration of 10% and the virus was stored at −80° C.until use. The virus was characterized by expression of the transgene,plaque forming units on 293 cells, particles/ml, endotoxin measurementsand PCR analysis of the virus and sequence analysis of the IL-22 codingregion in the virus.

A single dose of 5×10¹⁰ particles of recombinant adenovirus encodingmIL-22 was injected into the tail vein of female C57B1/6 mice, age 7-8weeks. Control mice received an adenovirus encoding GFP or PBS/10%glycerol. Mice from each experimental group were sacrificed at varioustime points post injection. For hematological and serum chemistryanalysis blood was collected by cardiac puncture. Blood was collectedvia retro-orbital sinus and differential counts were performed on bloodsmears. Tissue was harvested, fixed in formalin, and stained withhematoxylin and eosin for histopathology.

Example 4 Immunological Effects IL-22

The immunological effects of IL-22 were investigated in a metazoancontext by viral introduction of the cDNA of murine IL-22 into mice. Anadenoviral vector was used to express a cDNA of murine IL-22 in 8 weekold C57/B6 female mice by injection of 5×10¹⁰ viral particles eitherintravenously or subcutaneously. Test mice were sacrificed at 7 and 14days after injection and compared with control mice injected with bufferonly or with adenovirus expressing green fluorescent protein (GFP). Atdays 7 and 14, it was noted that the absolute and relative thymicweights were significantly decreased in the mice that expressed theviral murine IL-22. Absolute mean weight of the spleen was decreased onday 14 and liver weights were slightly increased on day 7. A grossgeneralized atrophy of the thymus as well as lymphoid depletion(observed microscopically) was apparent on days 7 and 14. An increase inkidney weight as well as enlargement of the liver were also observed.

In addition, there were a number of hematological effects that wereapparent on day 7, including decreased red blood cell count, hemoglobin,and hematocrit. These effects, taken together, indicated anemia in theanimals. Furthermore, there was an increase in platelets as well as anincrease in the white blood cell count due to an increase ofneutrophils. In light of these observations there was no evidence of aregenerative response, which indicated that the effects can be at thelevel of the bone marrow. A possible cause for this is the loss of smallmolecules through the kidney or gut. Furthermore, there was a slightdecrease in Albumin levels at day 7 and day 14 but an increase in serumamyloid A and fibrinogen levels, which are indicative of an acute phaseresponse. Other clinical observations included loss in body weight,signs of minimal dehydrations, increase urine specific gravity, adecrease in urine output and the induction of renal proximal tubularbasophilia. The basophilia observed is due to increased cell divisionand increased rRNA present in the epithelial cells of the renal proximaltube.

Example 5 Preparation and Characterization of Anti-IL-22 Monoclonal andPolyclonal Antibodies

Monoclonal and polyclonal antibodies were prepared using routinemethodologies also described in the instant specification. The tablepresented below illustrates the binding and neutralizing specificity ofmonoclonal antibodies P3/1, P3/2, P3/3 and P3/5 as well as chickenpolyclonal antibody that are directed against IL-22.

Rat Monoclonal Abs Polyclonal Ab IL-22 Antibodies P3/1 P3/2 P3/3 P3/5Chicken Polyclonal Binding Mouse Human Mouse/Human Mouse Mouse/HumanSpecificity Neutralizing Mouse Human Mouse/Human Mouse Mouse/HumanSpecificity

Binding specificity was determined by ELISA using mouse or human h/ftagged IL-22 microliter plates. Each antibody showed strong specificityfor either mouse or human IL-22. The neutralizing specificity wasdetermined by assessing the ability of the antibody to inhibit STAT 3phosphorylation mediated by 5 ng/ml mouse or human h/f tagged IL-22.Enzyme-linked immunosorbant assays (ELISA) using bound murine IL-22demonstrate that the mAb P3/1 has ˜5 nM ED₅₀ based on ˜2 nM for IL-22-Fcand ˜10 nM for H/F IL-22. Moreover, in addition to recognizingrecombinant IL-22, P3/1 mAb also binds native IL-22 secreted from Tcells that have been transfected with an IL-22 retroviral vector.

The IL-22 antibody P3/1 has been found to have an ID₅₀ of ˜1 nM, and towork stoichiometricly to block IL-22 activity when the cytokine ispresent at just saturating conditions (1 nM).

Example 6 Expression of IL-22 mRNA and Receptor

Expression of IL-22 and its receptor was examined semi-quantitativereverse-transcriptase polymerase chain reaction (RT-PCR) in a variety ofhuman and mouse tissues. The experiments reveal that IL-22 messenger RNA(mRNA) is present at very low levels in human testis, lung, prostate andperipheral blood lymphocytes (PBL) as normalized against control actin.Moreover, semi-quantitative RT-PCR shows that IL-22 receptor is detectedat highest levels in the human pancreas, and a lower levels in theliver, intestines, skin, thyroid, kidney, heart stomach, testis,salivary glands, adrenal glands and prostate. Alternatively, murineIL-22 receptor shows highest expression in the liver, small intestine,muscle, skin and ovaries, with lower expression in kidney and embryose8.5 and e19.

Example 7 In Situ Hybridization and Apoptotic Stain for IL-22 Protein

In situ hybridization for IL-22 protein and receptor messenger RNA(mRNA) of mice treated with adenovirus expression IL-22 (AdIL-22) orLipopolysaccharide (LPS) was performed and the results as follows:

A. Detection of IL-22 Cytokine mRNA

Tissue AdIL-22-treated mice LPS-treated mice Liver Day 1: staining incytoplasm of hepatocytes 6 hrs.: staining in cytoplasm of hepatocytesslightly positive slightly positive Days 3 and 14: no specific stainingSpleen Days 1, 3 and 14: slight staining in periarteriolar Negativeregion Heart N/A Negative Colon N/A Negative Kidneys Day 1: staining incytoplasm of proximal and 2 hrs.: staining in cytoplasm of proximal anddistal tubular epithelium, Henle's loop at distal tubular epithelium,Henle's loop at corticomedullary junction, parietal cells ofcorticomedullary junction was mildly positive Bowman space and someepithelial cells was 6 hrs.: staining in cytoplasm of the proximal andmildly positive distal tubular epithelium and Henle's loop at the Day 4:staining in cytoplasm of proximal and corticomedullary junction,glomerular tuft cells, distal tubular epithelium and Henle's loop atsome parietal cells of the Bowman space and corticomedullary junctionfew endothelial cells was slightly to moderately Day 14: staining incytoplasm of proximal positive tubular epithelium Pancreas N/A 2 and 6hrs.: staining in cytoplasm of acinar cells slightly positive Lungs N/A2 and 6 hrs.: staining in pneumocytes type II and/or intraaveolarmacrophages was slightly to mildly stained Stomach N/A 6 hrs.: stainingin cytoplasm of basal chief cells was mild Duodenum N/A 2 and 6 hrs.:staining in cytoplasm of enterocyte and brush border was moderate tomarked and Jejunum slightly positive in the intestine nervous plexuscells.B. Detection of the IL-22 Receptor mRNA in LPS-Treated Mice

Tissue LPS-treated mice Liver 2 and 6 hrs.: staining in the cytoplasm ofhepatocytes was slight to mild, nuclear staining was observed inheptocytes, bile duct epithelium and endothelial cells. Kidneys 2 and 6hrs.: staining was slight to moderate in the cytoplasm and nucleus ofproximal and distal tubular epithelium, Henle's loop at thecorticomedullary junction, glomerular tuft cells, some parietal cells ofBowman space and a few endothelial cells. Pancreas 2 and 6 hrs.:staining in cytoplasm of acinar cells slightly positive Heart 6 hrs.:nuclear staining was moderately positive in cardiomyocytes andendocardial and endothelial cells.

IL-22 receptor mRNA is additionally detected in small and largeintestine, stomach, lymph nodes, spleen, and lung. Expression of IL-22receptor can additionally be upregulated by a mediator of an innateimmune response, such as LPS.

Finally, TUNEL assays of kidney cells taken from c57BL/6 mice receivingmIL-22 protein intravenously showed a few apoptotic epithelial sells inseveral proximal convoluted tubules. Mice receiving saline intravenously(control group) demonstrated no positive staining.

These data demonstrate that both the cytokine and receptor can beinduced during an innate immune response, and that the induction isrestricted to tissues that are in an inflammatory state (LPS). During anadaptive immune response, IL-22 can also be induced from Th1 CD4+ Tcells. Since circulating leukocytes do not appear to have the receptor,this result suggests that IL-22 functions as an effector within tissuedownstream of an adaptive immune response, as is reinforced by thetissue expression of the receptor, constitutively and furtherupregulated by an innate inducer of inflammation.

Example 8 IL-22 Mediated Changes in Gene Expression

The ability of IL-22 to modulate levels of gene expression in livercells of mice infected with an AdIL-22 or Ad-GFP construct was examined.

Frozen mouse livers from day 1 and day 3 post-infection were pulverizedand RNA was purified using the Promega RNAgents Total RNA IsolationSystem (Promega, Madison, Wis.). The RNA was further purified using theRNeasy minikit. Total RNA was isolated from human PBMC's using theRNeasy minikit (Qiagen, Hidden, Germany).

Total RNA was prepared for hybridization by denaturing 10 μg of totalRNA for 10 minutes at 70° C. with 100 pM T7/T24-tagged oligo-dT primer(synthesized at Genetics Institute, Cambridge, Mass.), and cooled onice. First strand cDNA synthesis was performed under the followingbuffer conditions: 1× first strand buffer (Invitrogen Life Technologies,Carlsbad, Calif.), 10 mM DTT (GIBCO/Invitrogen), 500 μM of each dNTP(Invitrogen Life Technologies, Carlsbad, Calif.)), 400 units ofSuperscript RT II (Invitrogen Life Technologies) and 40 units RNAseinhibitor (Ambion, Austin, Tex.). The reaction proceeded at 47° C. for 1hour. Second strand cDNA was synthesized with the addition of thefollowing reagents at the final concentrations listed: 1× second strandbuffer (Invitrogen Life Technologies), an additional 200 μM of each dNTP(Invitrogen Life Technologies), 40 units of E. coli DNA polymerase I(Invitrogen Life Technologies), 2 units E. coli RNaseH (Invitrogen LifeTechnologies), and 10 units of E.coli DNA ligase. The reaction proceededat 15.80 C for 2 hours. During the last five minutes of the reaction 6units of T4 DNA polymerase (New England Biolabs, Beverly, Mass.) wasadded.

The resulting double stranded cDNA was purified with the use of BioMagcarboxyl terminated particles as follows: 0.2 mg of BioMag particles(Polysciences Inc., Warrington, Pa.) were equilibrated by washing threetimes with 0.5M EDTA and resuspended at a concentration of 22.2 mg/ml in0.5M EDTA. The double stranded cDNA reaction was diluted to a finalconcentration of 10% PEG/1.25M NaCl, and the bead suspension was addedto a final bead concentration of 0.614 mg/ml. The reaction was incubatedat room temperature for 10 minutes. The cDNA/bead complexes were washedwith 300 μl of 70% ethanol, the ethanol was removed and the tubes wereallowed to air dry. The cDNA was eluted with the addition of 20 μl of 10mM Tris-acetate, pH 7.8, incubated for 2.5 minutes and the cDNAcontaining supematate was removed.

10 μl of purified double stranded cDNA was added to an in vitrotranscription (IVT) solution which contained, 1×IVT buffer (Ambion,Austin, Tex.) 5,000 units T7 RNA polymerase (Epicentre Technologies,Madison, Wis.), 3 mM GTP, 1.5 mM ATP, 1.2 mM CTP and 1.2 mM UTP(Amersham/Pharmacia,), 0.4 mM each bio-16 UTP and bio-11 CTP (EnzoDiagnostics, Farmingdale, N.Y.), and 80 units RNase inhibitor (Ambion,Austin, Tex.). The reaction proceeded at 37° C. for 16 hours. LabeledRNA was purified with the use of an RNeasy (Qiagen). The RNA yield wasquantified by measuring absorbance at 260 nm.

12 μg of the in vitro transcription product was fragmented in 40 mMTris-actetate, pH 8.0, 100 mM potassium acetate, and 30 mM magnesiumacetate for 35 minutes at 94° C. The fragmented, labeled RNA probes werediluted in hybridization buffer at a final composition of 1×2-N-Morpholinoethanesulfonic acid (MES (buffer (pH 6.5), 50 μM Bio948(control biotinylated oligo that hybridizes to landmark features on theprobe array (Genetics Institute, Cambridge, Mass.), 100 μg/ml herringsperm DNA (Promega, Madison, Wis.), 500 μg/ml acetylated BSA (InvitrogenLife Technologies) and 1 μl/μg standard curve reagent (Proprietaryreagent supplied by Gene Logic, Gaithersburg, Md.). This hybridizationsolution was pre-hybridized with two glass beads (Fisher Scientific,Pittsburgh, Pa.) at 45° C. overnight. The hybridization solution wasremoved to a clean tube and heated for 1-2 min at 95° C. andmicrocentrifuged on high for 2 minutes to pellet insoluble debris.Oligonucleotide array cartridges (Murine 74Kκ2, Affymetrix, Santa Clara,Calif.) were pre-wet with non-stringent wash buffer (0.9M NaCl, 60 mMsodium phosphate, 6 mM EDTA and 0.01% Tween20) and incubated at 45° C.with rotation for 5-10 minutes. Buffer was removed from the cartridges,and the arrays were hybridized with 180 ul of the hybridization solutionat 45° C. rotating at 45-60 rpm overnight. After overnight incubationthe hybridization solutions were removed and the cartridges were filledwith non-stringent wash buffer. The array cartridges were washed using afluidics station according with 10 cycles of 2 mixes/cycle non-stringentwash buffer at 25° C. followed by 4 cycles of 15 mixes/cycle stringentwash buffer (100 mM MES, 0.1 M Na⁺, 0.01% Tween20 and 0.005% antifoam).The probe array was then first stained for 10 minutes at 25° C. in SAPEsolution (100 mM MES, 1M Na⁺, 0.05% Tween20, 0.005% antifoam, 2 mg/mlacetylated BSA (Invitrogen Life Technologies) and 10 ug/ml Rphycoerythrin streptavidin (Molecular. Probes, Eugene, Oreg.)). Afterfirst staining the probe array was washed for 10 cycles of 4 mixes/cyclewith non-stringent wash buffer at 25° C. The probe array was thenstained for 10 minutes at 25° C. in antibody solution (100 mM MES, 1MNa⁺, 0.05% Tween20, 0.005% antifoam, 2 mg/ml acetylated BSA (InvitrogenLife Technologies), 100 μg/ml Goat IgG (SIGMA, St. Louis, Mo.) and 3μg/ml biotinylated anti-streptavidin antibody(goat) (VectorLaboratories,). Following the second stain the probe array was stainedagain for an additional 10 minutes at 25° C. in SAPE solution. Finally,the probe array was washed for 15 cycles of 4 mixes/cycle withnon-stringent wash buffer at 30° C.

Arrays were scanned using an Affymetrix gene chip scanner (Affymetrix,Santa Clara, Calif.). The scanner contained a scanning confocalmicroscope and used an argon ion laser for the excitation source andemission is detected by a photomultiplier tube at 530 nm bandpass filter(fluorscein 0 or 560 longpass filter (phycoerythrin).

mRNA were analyzed on the Murine 74k (Mu74K) chip set. The data wasreduced with the use of GENECHIP 4.0 software. Each experimental samplewas compared to a time matched control in a two-file analysis. The datawere filtered with the criteria for genes that were called “Present” inone group, and removing all genes that were not called either“Increasing” or “Decreasing”.

Data for three mice are presented below (AD-GIL-19 Mouse 49, 51, and52). Shown are genes whose expression changed relative to Ad-GFPcontrol, with the indicated average-fold change shown for each animal.The changes observed in gene expression of Ad-IL-22 treated animals areconsistent with the induction by IL-22 of an acute phase response. Theobserved changes are also indicative of an inflammatory state in thetreated animal.

Day 3 Livers - U74v2 Ad-GIL- Ad-GIL- Ad-GIL- 19 Mouse 19 Mouse 19 Mousemouse number 49 51 52 Avg Fold Avg Fold Avg Fold Identifier Gene NameChange Change Change 1300017C10RIK RIKEN cDNA 23.4 17.2 19.3 1300017C10gene SAA-PS serum amyloid A, 24.6 13.9 24.3 pseudogene SAA1 serumamyloid A 1 11.9 9.7 12.3 SAA2 serum amyloid A 2 10.0 8.9 10.3 PRTN3proteinase 3 15.2 14.3 17.1 SPP1 secreted 10.2 7.8 10.7 phosphoprotein 1LCN2 lipocalin 2 13.4 10.3 13.3 SAA3 serum amyloid A 3 10.5 5.4 8.2 GRO1GRO1 oncogene 8.2 5.6 7.2 LY6D lymphocyte antigen 6 6.0 5.5 4.9 complex,locus D GRO1 GRO1 oncogene 7.0 5.6 7.2 RAD51L1 RAD51 like 1 (S. 4.4 3.73.8 cerevisiae) GAS6 growth arrest specific 4.1 3.5 4.8 6 SPI2-2 serineprotease 3.7 2.8 3.8 inhibitor 2-2 GADD45G growth arrest and 3.9 2.7 3.4DNA-damage- inducible 45 gamma CEBPD CCAAT/enhancer 5.3 3.2 3.9 bindingprotein (C/EBP), delta TNFRSF1A tumor necrosis factor 3.6 2.6 3.0receptor superfamily, member 1a CISH3 cytokine inducible 4.0 3.8SH2-containing protein 3 IL1R1 interleukin 1 receptor, 5.2 2.6 5.6 typeI SAP serum amyloid P- 3.1 2.5 3.3 component PEX11A peroxisomal 4.2 3.2biogenesis factor 11a 2310031E04RIK EST 2.9 2.7 3.3 AA880891 EST 2.7 2.42.8 CD14 CD14 antigen 3.4 2.3 2.6 MT1 metallothionein 1 2.7 2.4 2.9UNK_AW124835 EST 2.2 2.0 TM4SF7 transmembrane 4 2.6 2.8 2.4 superfamilymember 7 DNCLC1 dynein, cytoplasmic, 2.5 2.4 2.6 light chain 1 SAA4serum amyloid A 4 3.2 2.8 2410006H10RIK RIKEN cDNA 2.2 2.1 2.02410006H10 gene RBM3 RNA binding motif 2.7 2.8 2.8 protein 31300003D03RIK RIKEN cDNA 2.2 2.4 1300003D03 gene CEBPB CCAAT/enhancer2.0 2.3 binding protein (C/EBP), beta MT2 metallothionein 2 2.2 2.1 2.3ORM2 orosomucoid 2 1.7 1.7 2.0 VNN1 vanin 1 2.0 2.1 GTF2A2 generaltranscription 2.2 2.4 factor IIa, 2 (12 kD subunit) ITIH4 interalpha-trypsin 1.8 1.9 inhibitor, heavy chain 4 ITIH3 inter-alpha trypsin1.8 1.7 1.9 inhibitor, heavy chain 3 NPN3 neoplastic progression 2.2 2.53 U62673 EST −2.4 −3.2 PAPSS2 3′-phosphoadenosine −2.0 −2.35′-phosphosulfate synthase 2 TEMT thioether S- −2.2 −1.7methyltransferase TTR transthyretin −2.0 −1.8 CBG corticosteroid binding−3.4 −2.8 −2.8 globulin HSD11B1 hydroxysteroid 11-beta −2.1 −1.9dehydrogenase 1 LIFR leukemia inhibitory −2.5 −2.0 −1.7 factor receptorLIFR leukemia inhibitory −2.5 −2.0 −1.7 factor receptor HPGDhydroxyprostaglandin −1.9 −2.5 dehydrogenase 15 (NAD) CBG corticosteroidbinding −3.5 −2.8 −2.8 globulin HAL histidine ammonia −2.2 −2.0 −2.1lyase CYP2F2 cytochrome P450, 2f2 −2.5 −2.3 −1.7 KEG1 kidney expressed−2.9 −2.2 gene 1 AI266885 EST −4.7 −3.1 −2.4 Called Present in only oneanimal PAP pancreatitis- 9.2 associated protein 1300007C21RIK RIKEN cDNA4.7 1300007C21 gene REG2 rat regenerating islet- 9.8 derived, mousehomolog 2 UNK_AE000664 EST 9.6 SERINE/THREONINE- SERINE/THREONINE- 2.1PROTEIN KI . . . PROTEIN KI. 1300007C21RIK RIKEN cDNA 3.8 1300007C21gene CRAT carnitine 2.6 acetyltransferase AS2 arylsulfatase A 3.22310009M24RIK RIKEN cDNA 2.0 2310009M24 gene 2310004B05RIK RIKEN cDNA2.8 2310004B05 gene REG1 rat regenerating islet- 2.3 derived, mousehomolog 1 AW048468 esterase 31 1.8 PAP pancreatitis- 7.7 associatedprotein SULT-X1 sulfotransferase- −2.6 related protein SULT- X1 ES31esterase 31 −1.8 AW538652 EST −1.9 GAMT guanidinoacetate −2.0methyltransferase SC5D sterol-C5-desaturase −1.9 (fungal ERG3, delta-5-desaturase) homolog (S. cerevisae) GHR growth hormone −3.0 receptorAI839995 EST −1.8 0610025L15RIK RIKEN cDNA −1.9 0610025L15 gene AGXTalanine-glyoxylate −2.5 aminotransferases PAH phenylalanine −2.0hydroxylase IGFBP2 insulin-like growth −2.5 factor binding protein 2AI647632 EST −2.1 AI647632 EST −2.1 G6PC glucose-6- −2.2 phosphatase,catalytic CYP17 cytochrome P450, 17 −3.0 GSTA2 glutathione S- −2.3transferase, alpha 2 (Yc2) CYP26 cytochrome P450, 26, −9.0 retinoic acidretinoic acid THRSP thyroid hormone −2.7 responsive SPOT14 homolog(Rattus) FMO3 flavin containing −2.6 monooxygenase 3

Example 9 Effect of an Anti-IL-22 Antibody in an in Vivo Arthritis Model

The ability of the P3/1 monoclonal antibody to ameliorate symptoms in acollagen-induce arthritis (CIA) murine model was examined. Male DBA/1(Jackson Laboratories, Bar Harbor, Me.) mice were used for allexperiments. Antibody was administered prophylactically ortherapeutically to DBA mice. In the therapeutic regimen, treatment wasinitiated if disease was observed for two consecutive days in a mouse.

Arthritis was induced with the use of bovine collagen type II (Chondrex,Redmond, Wash.). Bovine collagen type II (Chondrex, Redmond, Wash.) wasdissolved in 0.1 M acetic acid and emulsified in an equal volume of CFA(Sigma) containing 1 mg/ml Mycobacterium tuberculosis (strain H37RA).100 μg of bovine collagen was injected subcutaneously in the base of thetail on day 0. On day 21, mice were injected subcutaneously, in the baseof the tail, with a solution containing 200 μg of bovine collagen in0.1M acetic acid that had been mixed with an equal volume of IncompleteFreund's adjuvant (Sigma). Naïve animals received the same sets ofinjections, minus collagen. The dosing protocol is shown schematicallyin FIG. 1.

Mice were monitored at least three times a week for disease progression.Individual limbs were assigned a clinical score based on the index:0=normal; P=prearthritic, characterized by focal erythema on the tips ofdigits.; 1=visible erythema accompanied by 1-2 swollen digits.;2=pronounced erythema, characterized by paw swelling and/or multi digitswelling.; 3=massive swelling extending into ankle or wrist joint.;4=difficulty in use of limb or joint rigidity. Thus, the sum of all limbscores for any given mouse yielded a maximum total body score of 16.

At various stages of disease, animals were euthanized, tissues wereharvested and paws were fixed in 10% formalin for histology or 4%paraformaldeyde, pH 7.47, decalcified in 20% EDTA (pH 8.0) and embeddedin paraffin for in situ hybridization. Using light microscopy the pawswere scored on a 5-grade scoring method (0-4) to characterize theintensity and extent of arthritis. Inflammatory infiltrates were usedfor scoring in addition to other changes related to the inflammation,such as pannus formation, fibrous of the synovial membrane, articularcartilage erosin and/or subchondral bone destruction. Histology gradeswere determined using readings of individual paws: NAD=0 or nothingabnormal discovered.; 1=Slight to moderate.; 2: Mild to moderate.; 3:Marked and 4:Massive.

The effect of the therapeutic administration of IL-22 antibody is shownin FIG. 2. Body score is shown as a function of time. Mice administeredanti-IL-22 antibody showed significantly decreased symptoms relative tomice administered control human IgG or PBS (data not shown).

The effect of prophylactic administration of neutralizing IL-22 antibodyis shown in FIGS. 3-5. Body score is shown as a function of timefollowing administration of anti-IL-22 or control antibody. Miceadministered anti-IL-22 antibody showed significantly decreased symptomsrelative to mice administered control rat IgG or PBS (data not shown).

Body score was also examined in mice subjected to a separateprophylactic regimen. The results are shown in FIG. 4 as a function oftime. Mice treated with control antibody demonstrated a significantlyhigher mean total body score than mice treated with anti-IL-22. iceadministered anti-IL-22 antibody showed significantly decreased symptomsrelative to mice administered control rat IgG1 or PBS (data not shown).

The progression of disease in paws of mice subjected to the prophylacticregimen is shown in FIG. 5. Mice at day 36 were sacrificed, and theseverity of disease in their paws examined. The paws were assigned ahistology grade of 0 to 4, with 0 corresponding to no disease and 4representing most severe disease. For rats injected with IL-22 antibody,over 60% had a histology grade of “0”, while about 20% of the mice had ahistology grade of “1”. About 15% of the mice showed a histology gradeof “2”, and about 10% of the mice showed a histology grade of “3”. Asmall percentage of mice showed a histology grade of “4”. For miceinjected with control antibody, about 30% showed a histology grade of“0”, and about 5% of the mice showed a histology grade of “1”. Theremaining mice exhibited more severe pathology grades: about 18% showeda histology grade of “2”, while 20% showed a pathology grade of “3”, andthe remaining mice showed a histology grade of “4”. Mice administeredanti-IL-22 antibody showed significantly decreased symptoms relative tomice administered control rat IgG 1 or PBS (data not shown).

These results demonstrate that administration of IL-22 antibody eitherprophylactically or therapeutically significantly ameliorates symptomsof arthritis in an animal system.

Example 10 In Situ Hybridization of IL-22 Transcripts

The expression of IL-22 and IL-22 receptor sequences in various celltypes of foot pads of arthritic mice was determined. Anti-sense IL-22and IL-22 murine receptor riboprobes were produced by generating 2independent PCR products from the corresponding transcripts. Theoligonucleotides 5′-GACTGATAATACGACTCACTATAGGGCGAACAATTTTGACTCCGATATTGTCCAAG-3′ (SEQ ID NO:6) and 5′-AGGATGGAGACATCTGACTGCCCTACG-3′ (SEQ IDNO:5) were used to generate for a IL-22 receptor sense probe and5′-ACAATTTTGACTCCGATATTGTCCAAG (SEQ ID NO:7) and3′-GACTGATAATACGACTCACTATAGGGCGAAGGATGGAGACATCTGACTGCC CTACG-3′ (SEQ IDNO:8) were used to generate for a IL-22 receptor antisense probe.Following PCR amplification probes were generated using T7 RNApolymerase and in vitro transcription.

A probe for IL-22 sequences was constructed by placing the followingsequence in a plasmid and placing the sequence under the control of T7and SP6 promoters to produce sense or anti-sense transcripts:

(SEQ ID NO: 9) CAGCCATACATCGTCAACCGCACCTTTATGCTGGCCAAGGAGGCCAGCCTTGCAGATAACAACACAGATGTCCGGCTCATCGGGGAGAAACTGTTCCGAGGAGTCAGTGCTAAGGATCAGTGCTACCTGATGAAGCAGGTGCTCAACTTCACCCTGGAAGACGTTCTGCTCCCCCAGTCAGACAGGTTCCA

T7 RNA polymerase binding sites were incorporated into theoligonucelotides to insert T7 binding sites at either the 5′end of thePCR product for sense riboprobe or the 3′end of the PCR product forantisense riboprobe. Digoxygenin labeled probes were prepared with theuse of a DlG RNA labeling mix (Roche Diagnostics, Mannheim, Germany), asdescribed by the manufacturer, and T7 RNA polymerase (RocheDiagnostics). IL-22 receptor mRNA-positive cells in the paw of CIAmurine model were macrophages, fibroblasts, a subpopulation oflymphocytes, activated osteoblasts, synoviocytes and epidermis. Nopositive staining was seen in the control paws or with sense probes.mIL-22 mRNA positive cells were: neutrophils, macrophages, fibroblastsand Osteocytes. No staining was seen in the paw section treated with thesense probe and the control mouse paw stained with mIL-22 mRNA. In situhybridization showed the presence of both the IL-22 receptor andcytokine in the paws of arthritic mice.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-33. (canceled)
 34. A method of treating an IL-22 related disordercomprising administering an IL-22 agonist in an amount effective to (1)modulate an IL-22 signal transduction pathway; (2) modulateproinflammatory cytokine production; (3) modulate lymphokine productionand/or secretion; (4) modulate production of adhesion molecules and/orcellular adhesion; (5) modulate expression or activity of nucleartranscription factors; (6) modulate secretion of IL-1; (7) compete withreceptors for other cytokines; (8) compete with another IL-22 familymember protein for binding to a IL-22 receptor; (9) modulate nucleartranslocation of internalized IL-22 receptor; (10) modulate cellularimmune responses; (11) modulate acute phase protein synthesis byhepatocytes, fever, or prostaglandin synthesis; and/or (12) promoteand/or potentiate wound healing.
 35. A method of protecting gut tissuein a patient comprising administering an effective amount IL-22 agonistto the patient.
 36. The method of claim 34 or 35, wherein the IL-22agonist is selected from the group consisting of human IL-22 protein;fragments, deletion mutants and addition mutants of human IL-22 protein;and peptide and small molecule compounds that interact with the receptoror other target to which human IL-22 is directed.
 37. The method ofclaim 34 or 35, wherein the IL-22 agonist is human IL-22 protein. 38.The method of claim 37, wherein the human IL-22 protein comprises aminoacids 1-179 of SEQ ID NO:2.